Copper nanoclusters (Cu NCs) have shown significant attention in sensing of molecular and ionic species. In this work, a single‐step biosynthetic approach was introduced for the preparation of fluorescent Cu NCs using Holarrhena pubescens (H. pubescens) leaves extract as a template. The synthesized H. pubescens–Cu NCs act as a nanomolecular probe for the detection of bilirubin in biofluids. The synthesized H. pubescens–Cu NCs displayed highest fluorescence intensity at 454 nm, when excited at 330 nm. Importantly, selective detection of bilirubin was obtained by introducing H. pubescens–Cu NCs as a simple molecular probe. The interaction of bilirubin and H. pubescens–Cu NCs resulted in a remarkable decrease in the emission peak intensity. The developed H. pubescens–Cu NCs‐based bilirubin molecular probe has a wide linear range of 0.5–20.00 μM with the limit of detection of 30.54 nM for bilirubin. The promising application of H. pubescens–Cu NCs‐based molecular probe was assessed by assaying bilirubin in spiked biofluids.