At his final news conference as ambassador, Locke said that Washington is "very concerned" about the case of Ilham Tohti as well as a recent increase in the arrests of social and legal activists and journalists.
Tohti is an economics professor and outspoken advocate for the Uighur Muslim minority who was arrested on Tuesday after being taken from his home one month ago.
China should value not just the economic welfare of its people, but also their freedom of speech, assembly and religion, Locke said.
"We believe that freedom of expression is a universal right and we very much are concerned about the arrest and detentions of people who are engaged in peaceful advocacy," Locke, accompanied by his wife Mona, told journalists at the U.S. Embassy in eastern Beijing.
State Department spokeswoman Jen Psaki on Wednesday also expressed deep concern for Tohti and called for his release.
China's authoritarian communist government brooks no political opposition and routinely rejects such remarks. Beijing says it must take harsh measures against what it calls Islamic radical terrorists fighting for the independence of the northwestern China Uighur homeland of Xinjiang amid an uptick in violent incidents over the past year.
Foreign Ministry spokeswoman Hua Chunying defended China's record at a regular briefing Wednesday and accused the country's critics of political bias.
Chinese citizens enjoy "unprecedented rights and liberty," Hua said. "We strongly oppose irresponsible comments made by anybody, regardless of which country he comes from."
A former commerce secretary and two-term governor of Washington state, Locke, 63, was the first Chinese-American to serve as ambassador to Beijing. Known for his affable, non-confrontational style, Locke placed a high priority on improving embassy efficiency and facilitating bilateral trade during his 2 1/2 years in Beijing at a time when exchanges are growing rapidly.
Locke said the wait for a visa interview by applicants in China had fallen from up to 100 days to as little as one, while visa applications grew by 75 percent. Almost 220,000 Chinese currently study at American colleges and universities, the most from any country.
Locke also oversaw the defusing of two of the most delicate diplomatic episodes between the countries in years.
In February 2012, Wang Lijun, the police chief in the western city of Chongqing, fled to a U.S. consulate in southwest China with information about the killing of a British businessman, setting off China's biggest political scandal in years. Wang's flight led to the removal and subsequent sentencing to life imprisonment for corruption of Chongqing's leader, Bo Xilai, formerly one of China's most powerful politicians.
Just two months later, blind legal activist Chen Guangcheng escaped house arrest and was given shelter in the U.S. Embassy in Beijing, where he remained for six days before being allowed to leave the country with his family to study in New York.
In his comments, Locke also repeated Washington's calls for restraint in China's maritime territorial disputes with Japan and its Southeast Asian neighbors. He also urged China to accord foreign journalists working in the country the same equitable treatment Chinese journalists receive in the West, a reference to Beijing's denial of visas to reporters from the New York Times and other news outlets.
Notably missing from his comments was any reference to Chinese investment barriers, alleged currency manipulation and other economic disputes that had been prominent themes in Locke's speeches earlier in his term. That appeared to reflect a strengthening U.S. economy and a boom in exports to China that are growing at nearly twice the rate they are to other countries. He said Chinese investment in the U.S. over the past two years exceed that of the previous 11 years combined.
"Mona and I depart China with a spirit of optimism. I believe the trajectory of our two countries' relationship is positive and indeed will shape the future of the world," Locke said.
Locke, a Democrat, said he had no plans for the immediate future but would stump for American political candidates and eventually return to China in a private capacity to work on business projects.
His replacement, former Montana Sen. Max Baucus, was sworn in last week and is expected to arrive within coming weeks.