The objective was to observe the influence of previously activated psoralens on the proliferation of K562 cells, and to provide laboratory data for its clinical usage. K562 cells were treated separately with previously and late activated psoralens, then their trypan blue exclusion inhibited rates (TBIR), cell proliferation inhibited rates (CPIR) and colony forming inhibited rates (CFIR) after culture were compared. The results showed that previously activated psoralens displayed an inhibiting effect on the proliferation of K562 cells with a dose-effect relationship. There was no obvious difference between previously and late activated psoralens on TBIR, CPIR and CFIR. In order to exert the inhibitive effect of previously activated psoralens, the time of ultraviolet ray exposure should be 10 minutes at least, and longer than 12 hours for inhibiting K562. The inhibitive effect of previously activated psoratens decreased as the time interval from activation to its use was prolonged. The inhibiting effect of previously activated psoralens was strongest within 6 hours after activation. In conclusion, both previously and late activated psoralens show inhibiting effects on the proliferation of K562, which may be able to use an antineoplastic drug in clinic.