This research presents the first novel green molecular-size-based fluorescence probe (MSBFP) as a spectroscopic strategy for detecting the Trimebutine drug. The method used a green, one-pot, direct spectrofluorimetric methodology to validate and assess the medication. Trimebutine drug and Cilefa Pink B formed an immediate ultra-fluorescent complex when mixed in an acidic environment. The fluorimetric study relied on Trimebutine's amplification of the dye response, which correlated to the generated complex's molecular size at 361 nm. Upon complexation, the molecular mass has grown from 504.5 to 1384.4 g mol−1. This growth is proportionally coupled to the drug concentration range of 0.035–1.5 µg mL−1. The lower and upper limits of the sensitivity varied from 0.010 and 0.029 µg mL−1, respectively. Trimebutine-Cilefa Pink B complexes were analyzed to determine optimal values for all the tunable system variables. Also, The International Council for Harmonization (ICH) requirements were successfully met by the system. In addition, this method effectively retrieved the drug in the intended pharmaceutical dosages. A significant achievement was using the developed fluorimetric method to monitor the drug of interest in human biofluids. The environmental friendliness of the planned procedure was then evaluated.