Complete degradation of lignocellulosic biomass to be useful products need complex enzyme. Cloning, expression and characterization of local recombinant β-xylosidase and cellulase have successfully produced in its heterologous host of Escherichia coli BL21. In this research, we cultured both of recombinant E. coli BL21 as enzyme cocktail in one flask at the same medium. Modified chemically defined medium (mCDM) was used as growth media, containing of natural extracts, minerals and salts. Modification also done by variation of mCDM with glucose, mCDM with glucose and IPTG, mCDM without glucose, mCDM without glucose but induced by IPTG. Determination of β-xylosidase and cellulase cocktails activities were done by commercial natural substrates of birchwood and carboxymethyl cellulose (CMC), and chromogenic substrates of p-nitrophenyl-β-xylopyranoside (pNP-X) and p-nitrophenyl-β-D-cellobioside (pNP-C). The result showed that enzyme cocktail of β-xylosidase and cellulase could produced by recombinant E. coli BL21 synergically. The highest activities on natural substrate of β-xylosidase was 0.834±0.018 U/mL and cellulase was 0.557±0.089 U/mL, produced in mCDM with glucose and IPTG. Further analysis showed β-xylosidase activity was 26.983±0.591 U/mL for pNP-X and cellulase was 33.777±1.083 U/mL for pNP-C. This result revealed the production of enzyme cocktail showed better results than compared to single enzyme. The synergism of β-xylosidase and cellulase as enzyme cocktails, provide effectiveness in the hydrolysis of biomass to be used as raw material for making environmentally friendly bioenergy.