A new, simple, and sensitive spectroscopic method has been developed for the determination of PMH in its pure form, in its pharmaceutical formulation, and in biological fluids (blood and urine) spectrophotometrically using copper nanoparticles (CuNPs) synthesized by the green method as a colorimetric detector without the use of chemical reagents. This method was characterized by being easy, sensitive, inexpensive, and environmentally friendly. A colored product (PMH-CuNPs) was formed. The absorbance was measured at the wavelength of 641.4 nm, and the linear range of (20–280) μg/ml, and it was found that the value of the correlation coefficient was (0.9983), the detection limit was (0.39) μg/ml, the quantitative limit was (1.18) µg/ml, and the molar absorption coefficient was (1315.69) L/mol. cm and Sandell significance were (0.2439) µg/cm², the average value of the percent recovery was 100.4%. The method was successfully applied to estimate Promethazine. HCL (PMH) in its pharmaceutical formulation and in biological fluids, and the characteristics of the colored product (PMH-CuNPs) were diagnosed. By measuring with several techniques, the average diameter was 73.54 nm in AFM technique, and the average crystal size through the XRD technique was 58 nm. SEM was measured, and the complex particles appeared in a spherical or semi-spherical shape, with an average size of 71.89, 82.04, and 79.25. FTIR was measured, and the functional groups were determined.