D-7-ACA (3-Ddeacetyl-7-amino cephalosporanic acid) is a new type of intermediate for cephalosporin antibiotics, which is used in the production of cefuroxime, cefdinir and other products with high yield and excellent quality. The content of D-7-ACA was determined by ultra performance liquid chromatography (UPLC) ，and calculated by the external standard method and according to peak area. The results showed that UPLC was well used to determine content of D-7-ACA with high specificity and very good linearity within 0.040 14～0.160 56 mg/mL, which correlation coefficient was 0.997 5，good precision and lower LOD and LOQ with 1.2×10-9and 4×10-9 respectively；the solution stability was long enough for 12 hours under cold storage condition, which RSD was 0.26%. The robustness was good thereby ensuring the detection results accurate when the chromatographic conditions changed, such as column temperature fluctuating 5 ℃, velocity of floating fluctuating 0.1 mL/min, mobile phase pH value fluctuating 0.1 units.