The invention discloses a method for measuring the amount of residual penicillin in milk. Standard working anhydrous alcohol solution with penicillin sodium is prepared, the pH value is regulated by dilute HCl to 2 plus or minus 0.5 or 4 plus or minus 0.5, the standard working anhydrous alcohol solution is bathed in boiling water, so that degradation products, i.e. penicillamine and penicillenic acid, are obtained, then the two degradation products respectively react with benzoquinone substance, so that charge-transfer complexes are obtained, and the working curves of different charge-transfer complexes are calculated. During measurement, the absorbance of the charge-transfer complex of milk sample is calculated by the same way, and is substituted into the working curve of the charge-transfer complex to work out the amount of the residual penicillin in the milk. The invention takes the lead in directly using the ultraviolet or fluorescent spectrophotometry to measure the amount of the residual penicillin in the milk. The method has the advantages of quick measurement, wide measurement range, high sensitivity, high accuracy and good selectivity, an expensive antibiotic detection instrument does not need to be purchased, the operation method is simple, and the method can reduce the production cost and the operation risk for diary processing enterprises, and therefore having a good application prospect.