The invention discloses a medicine effect evaluating method of a tumor multidrug resistance reversal medicine. According to the method of the invention, through detecting mass spectrum of a urine sample and/or serum sample in a health group, a negative control group, a positive control group and a to-be-evaluated medicine treatment group, the sample mass spectrum of the to-be-evaluated medicine treatment group is compared with the sample mass spectrum of the negative control group for calculating a peak area difference of the characteristic fragment ion peak of each metabolic marker in the sample of the to-be-evaluated medicine treatment group and the negative control group, evaluating the multidrug resistance of the to-be-evaluated medicine according to the difference. When the differenceis high, a callback value is high and the tumor multidrug resistance reversal effect is high. Through experiment verification, the method of the invention can accurately evaluate the medicine effectof the tumor multidrug resistance reversal medicine. Furthermore the method of the invention can be applied to early-period diagnosis for drug resistance of liver cancer cells.