An environment-friendly, sensitive, simple, and economic spectrofluorimetric method for the analysis of atenolol in pure powder, pharmaceutical preparations, and urine using green solvents was developed. The analysis was carried out using the solvent 0.02 N NaOH to reach the concentration range of 30-800 ng/ml, and fluorescence intensities were measured at 301±1nm after excitation at 255 nm using a slit width of 5 nm. The effects of different solvents, different pHs, and different concentrations of NaOH were studied. The method was applied successfully to pharmaceutical tablets and spiked urine samples. The proposed method was applied to investigate the urinary excretion pattern of atenolol in a healthy male volunteer after oral administration of a 50 mg single dose. Urine samples were collected at intervals for up to 24 hours. The method was validated according to the ICH guidelines, proving that it is accurate, specific, precise, and robust. So, it is suitable for quality control analysis of atenolol and for the detection of drug abuse in precision sports due to its sensitivity for the determination of atenolol on a regular basis at therapeutic urine levels. The method complies with the principles of green analytical chemistry due to its short analysis time, high sensitivity, low cost, simple instruments, small energy consumption, very little waste, and being free from harmful solvents, ensuring operator safety. The proposed method's high level of greenness was proved by its high eco-scale score (95 points) as well as by the GAPI and AGREE greenness assessments.