Abstract: [Objective] Biphenyl hydrolase (BphD) is an enzyme of the biphenyl biodegradation pathway in Rhodococcus sp. R04. Expression, purification and properties of BphD, together with chemical modification were investigated. [Method] Using heat-induced expression vector pBV220, we expressed bphD heterologously in E. coli BL21 (DE3). The products were purified by chromatography, and the purified enzyme was used to study its functions and properties by circular dichroism. [Results] BphD has been purified to homogeneity with a final purification fold of 5.02. With 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA ) as a substrate, the optimum pH and temperature of BphD are 9 and 80 ℃, respectively and the Km is 0.44 μmol/L. The enzyme had remaining 90% of activity incubating for 1 hour at 60 ℃, and had a half-life of 1 hour at 70 ℃, making it the most thermostable biphenyl hydrolase reported in Rhodococcus. Secondary structure of BphD has undergone great changes with the temperature up to 70 ℃, and has been severely damaged at 80 ℃. The sequence alignment between BphD and other homologous hydrolases as well as chemical modification of BphD indicated that Ser, His and Asp play important roles in the hydrolysis of HOPDA. In addition, Trp residue may be near the active center of enzyme as the key amino acid involved in substrate hydrolysis, which is not previously reported in the literature. [Conclusion] BphD with pH stability and thermostability is quite rare in the homologous hydrolases in Rhodococcus. The key status of Ser, His, Asp and Trp will build a basis for more learnings of the transformation mechanism of BphD.