Herein, a sensitive and selective spectrofluorimetric method has been developed for the determination of the ocular local anesthetic benoxinate hydrochloride (BEN-HCl) in eye drops and artificial aqueous humour. The proposed method is based on the interaction of fluorescamine with the primary amino group of BEN-HCl at room temperature. Following the excitation of the reaction product at 393 nm, the emitted relative fluorescence intensity (RFI) was measured at 483 nm. The key experimental parameters were carefully examined and optimized by adopting an analytical quality-by-design approach. The method used a two-level full factorial design (24FFD) to obtain the optimum RFI of the reaction product. The calibration curve was linear at the range of 0.10–1.0 μg/mL of BEN-HCl with sensitivity down to 0.015 μg/mL. The method was applied for analyzing the BEN-HCl eye drops and could also assess its spiked levels in artificial aqueous humour with high % recoveries (98.74–101.37%) and low SD values (≤ 1.11). To investigate the green profile of the proposed method, a greenness assessment was performed with the aid of the Analytical Eco-Scale Assessment (ESA) and GAPI. The developed method obtained a very high ESA rating score in addition to being sensitive, affordable, and environmentally sustainable. The proposed method was validated according to ICH guidelines.