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In this study, a post-selection strategy based on increasing key bases was proposed to improve aptamers' binding affinity. Phosphatidylserine (PS) was targeted as a model. Key bases of the original anti-PS aptamer were identified based on the molecular docking study, taken as repeating motif, and grafted onto the aptamer, with expectation to enhance the binding ability of the aptamer. An aptamer named PS-Apt-RKB2 was generated with significantly enhanced affinity. Biolayer interferometry assay results showed that the PS-Apt-RKB2 (Kd= 220.6 nM) exhibited a 29.59 times higher affinity over the original anti-PS aptamer (Kd= 6528 nM). Then, the PS-Apt-RKB2 was combined with chitosan oligosaccharide (COS) and AuNPs@Fe2+ to construct a label-free and COS-AuNPs@Fe2+ nanozyme-based colorimetric aptasensor. Under optimum conditions, the established aptasensor showed high sensitivity and demonstrated great application potentials in practice, with a limit of detection as low as 44.46 pM, and comparison rates of 91.21–105.4% when analyzing PS in real-world bioconversion samples with concentrations verified by high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This study can be further referred for developing well-performed aptamers and aptasensors for other targets. [Display omitted] • A post-selection strategy by increasing key base motifs was proposed. • A high affinity aptamer for phosphatidylserine (PS) was obtained with the strategy. • The aptamer showed a 29.59 times higher affinity than the original anti-PS aptamer. • A colorimetric aptasensor was developed with the aptamer and COS-AuNPs@Fe2+ nanozyme. • The aptasensor achieved highly sensitive detection of PS in bioconversion samples. [ABSTRACT FROM AUTHOR]