OBJECTIVE： To improve the detection method for the related substances of cyclophosphamide. METHODS： The TLC for the detection method of the cyclophosphamide related substances in Chinese Pharmacopoeia （2015 edition） was verified； the TLC was improved with references of compound A， B， C， D， the expanding agent of dichloromethane-acetic acid-methanol-water （50 ∶ 25 ∶ 15 ∶ 12，V/V/V/V） and the chromogenic agent of ninhydrin solution， and the detection test and methodology validation were conducted for the related substances. RESULTS： The original TLC can not detect the cyclophosphamide related compound B， C， D， and the operation process was cumbersome and not environmental protection. The detection limit of improved TLC for cyclophosphamide related compound A was 0.05 μg， and 0.1 μg for related compound B， C， D； specificity method test results showed that cyclophosphamide and the related compounds did not interfere with each other with a good separation. CONCLUSIONS： Improved TLC has the advantages of simplicity， environmental protection and high sensitivity， which can be used for the quality control of cyclophosphamide and its preparations.