The extracellular Aspergillus niger GZUF36 lipase (EXANL1) has broad applicability such as synthesis of functional lipids. The wild-type strain is unstable, and the culture time is long. Studies on the selectivity of substrates for EXANL1 are not available. Therefore, this study aimed to express, purify, characterize and analyze the substrate specificity of EAXNL1. The results showed that EXANL1 was stable in the presence of 40 % (v/v) dimethyl sulfoxide, glycerol, toluene, and n-hexane. The relative activity was 104 %, 88 %, 81 %, and 76 %. The residual enzyme activity of EXANL1 was more than 85 % in surfactants with a low concentration. EXANL1 exhibited a high conversion rate (79 %) for p-nitrophenyl palmitate. The molecular docking of p-nitrophenyl palmitate into the active site of EXANL1 indicated that the preference for this fatty acid chain length might be due to the formation of hydrogen bonds. The distance between the carbonyl carbon atom of p-nitrophenyl palmitate and the nucleophilic oxygen atom of serine 162 was 2.91 Å. Both experiments revealed that EXANL1 exhibited higher specificity for p-nitrophenyl palmitate. These results indicated that EXANL1 could be a promising tool in organic synthesis and detergent industry. The molecular docking provided a basis for the explanation on the substrate selectivity of EXANL1.