A bacterium, Pseudomonas putida SHI, which can catabolize phenol, naphthalene, or cresol as the sole carbon and energy source, was isolated from a petroleum-contaminated site in Taiwan. The catechol 2, 3-dioxygenase (C23O) was purified from this bacterial strain when grown on naphthalene as the sole carbon and energy source. The enzyme is composed of four identical subunits with a native molecular weight of 128 ± 5 kD. Small-angle neutron scattering (SANS) techniques were employed to study the thermal effects on the structural conformation of this enzyme in solution. The SANS measurements revealed distinct changes in the size of the enzyme between 50 and 80 °C, and the size was not restored during the subsequent cooling. The enzyme started to denature at 55 °C, and the structure was destroyed by the time the temperature reached 80 °C, at which the enzyme had become more than twice the original size. The optimal catalytic temperature of the enzyme was at 50 °C. The half-life of the activity at this temperature was 45 min. The enzyme activity increases starting from 25 °C and reaches its maximum at 50 °C, below which no obvious change in the size of the enzyme is found. Noticeable enlargement of the enzy me is revealed when the enzymatic activity starts to fall. By combination of SANS measurement and biochemical properties of the enzyme, this study demonstrates the correlation of enzyme size in solution and catalytic activity upon a heat treatment. In addition, for a protein composed of multiple subunits, the shape of the enzyme and the dissociation of the enzyme subunits in a thermal cycle were also demonstrated by SANS methodology.