The wide-spread usage of ciprofloxacin (CIP) resulted in its presence in different parts of the ecosystem. Thus, a simple, reliable, on-spot detection method for CIP is required in environmental context. Herein, a colorimetric assay is developed for the detection of CIP based on the branched polyethyleneimine (PEI) conjugated silver nanoparticles (PEI-AgNPs). AgNPs are prepared using PEI as stabilizing agent following a simple one-pot two-phase procedure. The prepared PEI-AgNPs are subsequently used for an efficient and selective detection of CIP. The characteristic yellow colour of PEI-AgNPs changed to colourless when CIP was added which was further confirmed by quenching in the intensity of the SPR (surface plasmon resonance) band (hypochromic shift). The proposed method is efficient for the quantitation of CIP in a linear dynamic range (LDR) of 0.1–200 µM with a limit of detection (LOD) of 0.038 µM, and limit of quantification (LOQ) of 0.12 µM. The developed method is selective, efficient, and sensitive to CIP in the presence of numerous interfering species and in real biological, environmental, and commercial pharmaceutical samples. Excellent performance of the proposed method compared to UV-Vis spectroscopy and UPLC in environmental, biological, and commercial pharmaceutical samples is demonstrated.