Zahra Shahid Hussain was the senior vice-president of Pakistan's Movement for Justice party (PTI), led by former international cricketer Imran Khan.
She was killed by gunmen on a motorcycle outside her home in the city's upmarket Defence neighbourhood.
Her murder comes on the eve of a highly-contested partial re-run of last Saturday's general election.
The reason for the shooting is unclear.
Imran Khan has blamed the city's dominant MQM party for her murder, a claim the party has strongly denied, BBC reports.
Pakistan's Dawn newspaper, citing police, said the shooting happened during an attempted robbery.
Local PTI leader Firdous Shamim told AFP news agency that Ms Hussain "was leaving her home for work when three gunmen attacked her. She thought they wanted to snatch her purse and handed it over to them but they killed her".
Hussain was reportedly rushed to hospital but succumbed to her injuries on the way.
Pakistani President Asif Ali Zardari strongly condemned the murder, describing it as a "tragic incident".
Sunday's partial re-run of the vote in Karachi was ordered after Mr Khan's party accused the MQM of widespread vote-rigging and intimidation.
Voice of Russia, BBC