Herein, a novel biomaterial of crosslink chitosan-glutaraldehyde combined with Bacillus subtilis biomass (CHS-GLU/BCL) was synthesized via hydrothermal synthesis to effectively remove the anionic dye (Acid red 88; AR88) from synthetic wastewater solution. The features of the CHS-GLU/BCL biomaterial were assessed using SEM-EDX, pHpzc, BET, and FTIR studies. To optimize the adsorption efficiency of CHS-GLU/BCL to remove AR88 dye a Box-Behnken design (BBD) was utilized. The experiment utilized three independent variables including, the dosage of CHS-GLU/BCL (A: 0.02–0.1 g/100 mL), the contact time (B: 5–30 min), and the pH of the AR88 solution (C: 4–10). The investigation of adsorption kinetics confirms that the AR88 dye adsorption onto the CHS-GLU/BCL biomaterial follows the pseudo second order and pseudo first order kinetics models. Moreover, the isotherm data fits the Langmuir isotherm model. The maximum adsorption capacity (qmax) of AR88 dye onto CHS-GLU/BCL biomaterial was determined to be 148 mg/g in an acidic pH environment (pH = 4). The adsorption mechanism of AR88 and dye onto the biomaterial surface can be related to many connections, such as hydrogen bonding, n-π interactions, and electrostatic attraction. Hence, the biomaterial CHS-GLU/BCL has a good potency affinity for adsorbing anionic dye.