Antimony (Sb) is one of the primary pollutants and its compounds and therefore its removal from wastewater and natural waters is significant using effective and eco-friendly adsorbents. Graphene nanoplatelets (GNP) are an important graphene derivative with a varying number of exfoliation cycles which is referred to as multi-layered or few-layered graphene. In this study, Staphylococcus aureus (S. aureus) was immobilized with GNP for adsorption of Sb(III) from the aquatic system. The batch study conditions, composite dosage (100–300 mg/L), Sb(III) initial concentration (1–300 mg/L), pH level (2–8), contact time (10–50 min), and temperature (24–60 °C) were optimized. The adsorption efficiency of the composite was also investigated for wastewater samples including ions K+, Cl−, Na+, Mg2+, Ca2+, NO3−, PO43−, SO42-, and several heavy metal ions. The factorial design is broadly utilized to estimate the statistical correlation among experimental variables and responses. The equilibrium data were studied for Langmuir and Freundlich isotherms under optimized adsorption conditions and the Langmuir adsorption capacity of (qm) of GNP/S. aureus composite was estimated as 116.1 mg g−1. The adsorption kinetic mechanism well followed the pseudo-second order model with a non-linear correlation coefficient (R2 = 0.999). The electrostatic, π–π interactions, and hydrogen bonding interactions as well as surface adsorption make feasible removal of Sb(III) ions onto GNP/S. aureus composite sorbent. The prepared GNP/S. aureus composite is a promising adsorbent to remove Sb(III) ions from aquatic systems.