Mitoxantrone (MXN) is a synthetic anthracenedione oncogenic therapy. It is often prescribed as an anticancer agent to manage a variety of cancers. A green, fast, and easy fluorimetric technique for the assay of MXN as a topoisomerase type II enzyme suppressor. An investigation of MXN's fluorescence behavior in various media and solvents constituted the basis for this new technique. Methanol was shown to enhance the intrinsic fluorescence considerably. After excitation at 610 nm, the highest fluorescence intensity was found at 675 nm. Various experimental parameters, such as media, solvents, and pH levels, were tested and adjusted. ICH (International Conference on Harmonization) guidelines were followed when validating procedures. It was possible to achieve linearity in the 0.02–1.50 μg ml−1 with the method. The sensitivity (in terms of limit of detection and limit of quantification) was 0.003 and 0.008 μg ml−1, indicating low toxicity. As a result, the current technology has a remarkable recovery for detecting residues in diverse bodily fluids. Also, the quantum yield was estimated for the designed system. Finally, the method was rated by eco‐scale scoring.