Synthesis of coumarin derivative from the condensation of benzocoumarin-4-ethylacetate with salicylaldehyde hydrazone has been used as fluorescent chemo-sensor. The synthesized compounds have been characterized using FTIR, UV-vis, 1HNMR and Mass spectroscopy. Solvent effects on the excitation and emission spectra of the synthesized benzocoumarin hydrazide have been studied. A simple, rapid, sensitive, accurate and precise Spectrofluorimetric method has been developed for assessment the concentration of Nalbuphine Hydrochloride (NAL) in human serum samples. The suggested method is based on measuring the enhancement of the fluorescence intensities of the new synthesized benzocoumarin hydrazide (BCH) as a fluorescence chemical sensor at λem 415nm using λex 340nm. The fluorescence intensities- concentration plot of NAL was linear over the range (3.6×10−8–4.9×10−6molL−1) with a lower detection limit (LOD) of 8.69×10−8molL−1 and lower quantification (LOQ) of 2.63×10−7molL−1. The validity and success of the proposed spectrofluorimetric method in the present work for assessment the concentrations of NAL in serum samples with an average percent recovery ±RSD% of 100.48±1.88. The calculated Bias of the percent relative error (%RE) is 5.8 which confirmed the high accuracy and precision of the proposed method. Molecular docking studies using MOE have been carried out to predict the binding modes between the ligands and active sites of the Oxidoreductase [Nitric Oxide Synthase EC# (1.14.13.39) PDB ID: 2BHJ and Amine Oxidase B EC# (1.4.3.4) PDB ID: 2V60], hydrolase [Proteinase K EC# (3.4.21.64) PDB ID: 2PWB] and Lyase [Carbonic anhydrase 2 EC# (4.2.1.1) PDB ID: 3IEO] enzymes.The observed activity of BCH and BCA Compounds gave rise to the conclusion that they might exert their actions through inhibition of the Oxidoreductase enzymes.