The research on fungal α-amylase mainly focused on the biochemical properties such as the substrate catalytic mechanism, and the relationship between the composition of amino acids residues and the functional properties of this enzyme is unclear. In this paper, three special conserved histidine residues (H120, H200 and H286) that existed in the catalytic domain of Rhizopus oryzae α-amylase were identified by bioinformatics analysis methods of molecular docking and comparative analysis. A series of mutants were constructed base on the above three identified sites using site-directed mutagenesis. The results showed that the structure of the amino acid residue at position 286 has been proved to be related with the optimum temperature, optimum pH, acid resistance and maltose-forming ability of the enzyme. The acid resistance and maltose-forming ability on soluble starch of the mutant H286L were significantly improved. Changes in 120 and 200 amino acid residues exhibited no effects on the optimal temperature, optimum pH and maltose-forming ability, but the combined mutations at both sites of 120 and 200 could improve its acid resistance. The resulted data indicated that the high maltose-forming ability of the enzyme was not directly related to its affinity on maltotriose, which may provide a theoretical reference for the mechanism of high maltose-forming ability and its directed evolution for fungal α-amylase.