Development of simple, facile, and on-spot colorimetric assays for drugs and pesticides based on metal nano -particles is an emerging field of modern scientific research. Herein, a branched polyethyleneimine (PEI) is used to stabilize silver nanoparticles (AgNPs) through a simple one-pot, two-phase protocol. As the primary goal of the research, the synthesized PEI-AgNPs are employed for an efficient and selective quantitative detection of promethazine (PRO). The typical yellow color of PEI-AgNPs changed to orangish-brown as soon as PRO is added which is further endorsed by an increase in the intensity of the SPR band. The electrostatic forces are responsible for the steric stabilization of silver ions in the micro-domains of the PEI. PRO addition in PEI-AgNPs decreased the average hydrodynamic size and increased apparent positive charge of AgNPs. These changes might be attributed to the presence of the sulfur group in PRO. The limit of detection, the limit of quantification, and the dynamic range of detection of PRO through PEI-AgNPs are found to be 0.003 mu M, 0.1 mu M, and 0.1-200 mu M, respectively. Moreover, PEI-AgNPs allowed reliable, efficient, selective, and quick quantitative detection of PRO in environmental, biological, and commercial drug samples in the presence of other interfering drugs and metal salts. The proposed PRO sensor renders an excellent platform for its quick and on-spot detection without requiring a well-equipped lab setup. Up to the best of our knowledge, the proposed PRO sensor is the first of its kind (colorimetric) in the list of proposed instrumental methods for determination of PRO. The quantitation of PRO as obtained by proposed sensor is compared with UV-Vis spectroscopy and UPLC. Finally, excellent performance of PEI-AgNPs based sensor for biological, environmental, and commercial samples is demonstrated in comparison to UV-Vis spectroscopy and UPLC.