The regulations were brought in after mass pro-democracy protests in 2019 with Beijing saying it would bring stability to the region but critics say it has been used to silence dissent.
Some of those ordered to report to the police helped run an unofficial “primary” election last June to pick opposition candidates for 2020 elections which were then postponed.
Hong Kong police said in a statement: “Police this afternoon laid a charge against 47 persons...with one count of ‘conspiracy to commit subversion’.”
The 39 men and eight women, aged between 23 and 64, are scheduled to appear before West Kowloon Magistracy on Monday.
Among them is journalist-turned activist Gwyneth Ho who previously described the national security law as an attempt “to silence the political voices in Hong Kong”
Earlier this month, Foreign Secretary Dominic Raab put diplomatic pressure on the United Nations to respond to China’s “appalling treatment” of people in Hong Kong.
It comes amid heightened tensions between Britain and China after Beijing banned BBC World News in retaliation after broadcast regulator Ofcom stripped state TV channel China Global Television Network of its UK broadcasting licence.
The UK last year also banned technology giant Huawei from being used in the country’s 5G communications network out of fears it could be used by the Chinese government to spy on Britain.
Addressing the UN Human Rights Council, he said “no-one can ignore the evidence any more” of a deteriorating human rights situation in China and called for international action.
In his online speech, Mr Raab said people’s rights in Hong Kong are being “systematically violated” and that the national security law is a “clear breach of the Sino-British Joint Declaration” that is having a “chilling effect on personal freedoms”.
“Free and fair legislative elections must take place, with a range of opposition voices allowed to take part,” he urged.