Objective To obtain the finest aptamers with stronger affinity and shortest sequences from the combination of molecular docking simulation and surface plasmon resonance (SPR) evaluation experimentation.Methods Towards three previously screened single-stranded DNA aptamers against ricin with 80 nt, L14, P3, L7, on the confirmation basis of binding ability between their 2D stem-loop sequences of random region with target proteins, the H-DOCK molecular docking was performed to guide the determination of their minimum aptamer binding motifs (MABM), and an elongated stepping sequence cluster was sequentially constructed. The affinity and kinetic parameters of the designed clusters were measured by SPR, in which the binding key structures of the selected aptamers were depicted, and finally the finest aptamers could be selected.Results All three random region aptamers, L14r, P3r and L7r, can form some certain hairpin structures, and the affinity of L14r is increased by nine times than L14, L7r increased by 2 times, and P3r kept the same. The results of molecular docking between the random region aptamers and ricin showed that, the docking scores of L14r, P3r and L7r were all lower than negative sequence of 40T, the number of key binding amino acids were 11, 8 and 9, and the predicted binding sites with distances less than 5 ? were 20, 12 and 15, respectively, indicating good binding ability with ricin. Further, the sequence composition of MABM, L14rm, P3rm and L7rm, were deduced from the binding structures confined in the ricin active pockets, and the elongated stepping sequence cluster was built. On the parameters including the number of key binding amino acids, binding sites, the docking scores, as well as the results of SPR evaluation, the finest aptamers were evolved, in which the affinity of L14rm and L7rm-2 continued to be increased by 1-2 times.Conclusion The random region aptamer can effectively bind ricin with stronger affinity than the full-length aptamer. Molecular docking and stepping sequence clusters design can aid the fast evolution of three finest aptamers from only 17 sequences as well as the investigation of binding interaction. The K_D values of the three finest aptamers against ricin, L14rm, P3r and L7rm-2, were (64±30), (167±19) and (120±1) nmol/L, respectively, and the affinity was increased to 14, 1 and 4 times of the full-length aptamers.