Objective：To determine whether the key glycolytic enzyme enolase（Eno） bind heat shock protein DnaJ directly and to analyze the binding sites between DnaJ and Eno in Streptococcus pneumoniae（S.pneumoniae）. Methods：Recombinant Eno protein was obtained through prokaryotic expression system，and its α-enolase activity was analyzed. Kunming mice were immunized with the recombinant protein to prepare the polyclonal antibody to Eno protein and the titer of the antibody was determined by ELISA. The expression levels of Eno protein in S.pneumoniae with different serotypes were determined by Western blot. The direct interaction between DnaJ and Eno was determined by biolayer interferometrvy（BLI） technology and direct binding assay. Truncated Eno protein was expressed and purified，and the binding sites of DnaJ on Eno were determined by direct binding assay. Results：The recombinant protein Eno with high purity（about 90%） was obtained after purification of Ni affinity chromatography and exhibited α-enolase activity. The antibody titer of anti-Eno in the immunized mice serum reached to 7.68×106. Western blot showed the same band with Mr 47 000 presented in the supernatant of all the 7 serotypes of S.pneumoniae. The direct binding assay demonstrated that DnaJ bound Eno in a dose-dependent manner（r=0.920，P=0.000） and a concentration dependent binding of Eno to immobilized DnaJ was detected（KD=926 nmol/L） by BLI. Binding experiments demonstrated that DnaJ bound more truncated Eno（aa1-aa100）（2.25±0.38） than the other truncated Eno proteins（0.94±0.25），（1.08±0.09），（0.75±0.12）（P=0.000，P=0.001，P=0.000）. Conclusion：DnaJ interacts with Eno directly in S.pneumoniae and primarily via the binding sites，which are localized within 100 aa of the N-terminal domain of Eno.