Exploitation of rhizosphere inhabiting Pseudomonas fluorescens for useful products like industrial enzymes remains largely less explored. Glucanase enzyme produced by P. fluorescens has wide range of application in food, textile and brewing industry, other than their antifungal activity for agricultural use. In our study, native isolates of P. fluorescens from rice and sunhemp rhizosphere varied in their preference to carbon and nitrogen source nutrients under in vitro conditions attributing to the possible difference in the metabolic profile of their respective ecological niche. The optimum conditions for maximum growth and glucanase enzyme production were a pH of 6.5 for both isolates and temperature of 37 and 40°C for rice and sunhemp isolates respectively. The rice rhizosphere isolate of the bacteria was a rapid producer of glucanase (24 h) compared to sunhemp isolate (48 h). Extracellularly secreted proteins were separated and partially purified by dialysis and sephadex column chromatography. Electrophoretic resolution of purified proteins in correlation with their enzyme activity revealed a 29 kDa isoform of glucanase in rice isolate and a 32 kDa isoform of the same in sunhemp isolate. In-gel activity assay was also performed to confirm the identity and activity of the purified protein. Key words: Glucanase, Pseudomonas fluorescens, purification, rice, sun hemp.