This work reported a green method of synthesizing silver nanoparticles (AgNPs) with bagasse acting as reducing and stabilizing agents to rapidly detect l-Cysteine (Cys). The optimal condition of synthesizing AgNPs was obtained by varying the reactant ratio, temperature, and reaction time. The obtained AgNPs with diameters of 18–28 nm were almost spherical and capped by bagasse extract (BAE), and even they were redispersed well in water after centrifugation. The content of Ag in BAE/AgNPs composite was high up to 45.4%. The possible forming mechanism of AgNPs was investigated. Importantly, the BAE/AgNPs composite showed highly sensitive detection of Cys with a limit of detection (LOD) of 35 nM and selective detection of Cys among 19 amino acids. Besides, the BAE/AgNPs composite was applied in Cys detection in human serum with the error less than 5%. Thus, this work provided a rapid method for Cys detection via green-synthesized AgNPs, and the synthesis method provided a direction for recycle of nanometal.