Introduction: Microcrystalline cellulose is a cellulase derivative which usually used as a pharmaceutical excipient in the manufacturing of direct compression tablet. High concentration of cellulose is found in water hyacinth (about 60%) therefore it might be used in the manufacturing of microcrystalline cellulose. This study was aimed to obtain the best cellulolytic fungi, the best optimal conditions of enzymatic hydrolysis and comparing microcrystalline cellulose characteristics obtained from water hyacinth with microcrystalline cellulose standard, Avicel pH 101. Method: This study began with isolation of cellulolytic fungi, and then enzymatic hydrolysis of α-cellulose was done with cellulase enzymes extracted from cellulolytic fungi with various pH, temperature, duration and enzyme concentration. Microcrystalline obtained was characterized by XRD (X-Ray Diffraction) and then the result was compared with Avicel pH 101. Results: The results showed the best cellulolytic isolate was isolate 2 and its optimal hydrolysis conditions at pH 5, 30⁰C, for 1 h with 5 mL (5% v/v) enzyme. Based on the pattern of diffraction there was a similarity between microcrystalline cellulose of enzymatic hydrolysis result compared with Avicel pH 101.