A method for the simultaneous spectrofluorometric determination of naproxen drug enantiomers has been proposed. The method is based on three procedures. These procedures have their precision limitation which has been fully investigated and the optimized condition has been proposed. In this regard, magnetite nanospheres have been chemically modified with L-(−)-tryptophan (Try-MNSs). The synthesized nanospheres have been characterized by FT-IR, XRD, VSM and TEM measurements. The results showed that Try-MNSs have good enantioselectivity toward S-(+)-Naproxen and could efficiently remove it in the presence of its enantiomer. Using the proposed procedures, precise naproxen drug enantiomeric composition analysis in a concentration range of 100.0–1800.0 ng mL⁻¹ is achievable. Furthermore, S-(+)-Naproxen determination in a 2.0–50.0 ng mL⁻¹ concentration range with a detection limit of 0.7 ng mL⁻¹ is feasible.