Objective The purpose of this study is to improve the affinity and selectivity of aptamers of progesterone (P4).Methods The computer-aided optimization strategy (in silico maturation, ISM) based on genetic algorithm (GA) was used. The 4 rounds of GA (including crossover mutation, single point mutation and double point mutation operations) were performed to construct the initial library and G1, G2 and G3 generation ssDNA oligonucleotides as new candidate aptamer libraries. The candidate aptamers were isolated and analyzed by molecular docking, and continuously optimized using an iterative strategy. In addition, a new strategy is proposed to predict the tertiary structure of ssDNA more accurately. Their secondary and tertiary structures were modeled by Mfold and RNA composer respectively. Output PDB files were modified from RNA to DNA in the Discovery Studio software. Finally, the structure is energy minimized by Molecular Operating Environment.Results In the second generation, P4S-0 sequence was optimized in the local search space, and P4G1-14, P4G2-20, P4G1-6, P4G1-7 and P4G2-14 were selected as the best aptamers for P4. AuNPs colorimetric method was applied to verify the affinity of the optimized aptamer. And then a fluorescence method based on the structure switch of the aptamer was constructed to determine the dissociation constant (K_D) of the aptamer, and the selectivity of the aptamer (for bisphenol A, estradiol, testosterone and cortisol) was evaluated.Conclusion The aptamer optimized by ISM has a greater affinity for P4 than the original aptamer, and still retains the selectivity for recognizing molecules with similar structure.