[Objective] To efficiently express and purify wild-type and three active site-deleted variants (ENO1-M1, ENO1-M2, and ENO1-M3) of ENO1 through the insect cell/baculovirus expression system, thus providing a theoretical basis for subsequent functional research, antibody development, and inhibitor screening. [Methods] Wild-type and active site-deleted genes of ENO1 with a C-terminal 6×His tag were cloned into the pFastBac-HTB vector via molecular cloning and then transformed into DH10Bac competent cells to obtain recombinant Bacmid through screening. Recombinant baculovirus particles were transfected into ExpiSf9 insect cells to generate the P0 virus stock, which was then amplified to produce high-titer P1 virus for target protein expression. Expression products were purified by affinity chromatography (e.g., nickel column) and identified by SDS-PAGE, Coomassie Brilliant Blue staining, and Western blotting. The anti-ENO1 monoclonal antibody, single-chain antibody, and chimeric antibody were used for specific detection by Western blotting. On the basis of the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) under the catalysis by ENO1, the specific activity of ENO1 was measured by UV spectrophotometry at 240 nm. [Results] All recombinant plasmids and bacmids were successfully constructed, and high-level expression of recombinant proteins was achieved in insect cells. High-purity recombinant proteins were obtained, and Western blotting analysis confirmed their reactivity. The deletion of active sites of the enzyme did not affect the immunoreactivity. The specific activity of wild-type ENO1 was 691.28 U/mg, while those of the three deletion variants were significantly reduced. ENO1-M2 (with deletion of GSHAGNK at residues 156-162) and ENO1-M3 (with deletion of SPDPSRYI at residues 262-270) exhibited particularly pronounced losses of enzyme activity. [Conclusion] We successfully expressed and purified both wild-type and active site-deleted ENO1 proteins with retained reactivity by using the baculovirus expression vector system. The results confirm that GSHAGNK and SPDPSRYI are two key active sites of this enzyme, laying both theoretical and material foundations for further exploring the functions of ENO1 and developing its specific inhibitors.