The effectiveness of NaOH-functionalized Aspergillus niger-filled sodium alginate microbeads on methylene blue (MB) removal was investigated in this study. First of all, Aspergillus niger biomass was obtained, then functionalized using sodium hydroxide (NaOH). Lastly, the composites were prepared by dispersing NaOH-treated-Aspergillus niger in sodium alginate gel. The cross-linked composite microbeads in a 3% CaCl2 solution were characterized by Fourier-transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, atomic force microscope (AFM), and scanning electron microscope (SEM). FTIR-ATR results indicated that the hydroxyl, amine, and carboxyl groups on the surface of the composite beads acted as binding sites. It was determined that the biosorption of MB occurred via the electrostatic interactions, Van der Waals interactions, pi-pi interactions, and H-bonding between functional groups and dye molecules. SEM and AFM analyses proved the presence of adsorbed dye molecules on the surface. Besides, optimum conditions were determined by examining the effects of different physicochemical parameters on biosorption. The maximum biosorption capacity of microbeads was obtained as 322.6 mg/g in pH = 7 at 25 degrees C. Moreover, various isotherms were used, including Freundlich, Langmuir, Dubinin-Radushkevich, and Harkins-Jura. The Langmuir model was found to be the best feasible when the correlation coefficients were taken into account. Biosorption followed the pseudo-second-order kinetic and occurred exothermically and spontaneously. This study indicates that NaOH-treated Aspergillus niger-filled sodium alginate microbeads are promising material as a biosorbent.