Sensitive monitoring of the target products during the biosynthesis process is crucial, and facile analytical approaches are urgently needed. Herein, phosphatidylserine (PS) was chosen as the model target, a colorimetric aptasensor was developed for the rapid quantitation in biosynthesis samples. A chimeric aptamer was constructed with two homogeneous original PS aptamers. Specific recognition between the chimeric aptamer and PS results in the desorption of aptamer from the surface of the AuNPs nanozyme, and the peroxidase-like enzymatic activity of the AuNPs nanozyme was weakened in a relationship with the different concentrations. The developed aptasensor performed well when applied for analyzing PS in biosynthesis samples. The aptasensor offers good sensitivity and selectivity, under optimal conditions, achieving monitoring and quantitation of PS in the range of 2.5–80.0 μmol/L, with a limit of detection at 536.2 nmol/L. Moreover, the aptasensor provides good accuracy, with comparison rates of 98.17%–106.40%, when compared with the HPLC-ELSD. This study provides a good reference for monitoring other biosynthesized products and promoting the development of aptamers and aptasensors in real-world applications.