The announcement came a day after North Korea warned of pre-emptive nuclear strikes in response to the start of US-South Korean military drills it views as a rehearsal for invasion. This year's annual drills, set to run until late April, are the largest ever.
The South Korean sanctions target 38 individuals and 24 organisations in North Korea who Seoul says are responsible for the North's development of weapons of mass destruction.
It said South Koreans will be barred from engaging in financial and foreign exchange dealings with the blacklisted people and organisations, whose asserts in South Korea will be frozen.
South Korea also said it will ban the entrance of any ship that has stopped at a North Korean port in the previous 180 days. Currently only North Korean ships are banned.
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It said it will continue to advise South Korean citizens not to eat at North Korea-run restaurants around the world. North Korea operates about 130 restaurants in China and other countries.
The UN Security Council last week adopted its toughest sanctions against North Korea in two decades. North Korea described them as "nothing new" but warned it would take unspecified "merciless" steps in response.
South Korea's announcement underscores its pursuit of a hard-line policy toward the North. South Korea earlier halted operations at a jointly run factory park in North Korea, the last major cooperation project between the rivals.
The North Korean entities targeted by the new sanctions include banks, trading and shipping companies, and an atomic research center.
Many of the North Korean individuals and entities are already under US, Japanese, UN or other international sanctions.
Earlier today, Seoul's spy agency accused North Korea of having hacked into the smartphones of dozens of key South Korean officials, stealing text and voice messages and their phone call logs.