Monitoring therapeutic drugs and their elimination is crucial because they may cause severe side effects on the human body. Methotrexate (MTX) is a widely used anti-cancer drug, which is highly expensive, and the detection of unwanted overdoses of MTX using traditional procedures is time-consuming and involves complex instrumentation. In this work, we have developed a nanocomposite material using phosphorene, cystine, and gold (Ph-Cys-Au) that shows excellent optical properties. This nanocomposite can be used as an optical sensing platform for the detection of MTX in the range 0-260 μM. The synthesized sensing platform is very sensitive, selective, and cost-effective for the detection of MTX. Ph-Cys-Au can effectively detect MTX in aqueous media with a limit of detection (LOD) of about 0.0266 nM (for a linear range of 0-140 μM) and 0.0077 nM (for a linear range of 160-260 μM). The nanocomposite is equally selective for real samples, such as human blood serum (HBS) and artificial urine (AU) with a LOD of 0.0914 nM and 0.0734 nM, respectively. We have also determined the limit of quantification (LOQ); the LOQ values for the aqueous media were 0.0807 nM (for a linear range of 0-140 μM) and 0.0234 nM (for a linear range of 160-260 μM), whereas, the values for HBS and AU were around 0.2771 nM and 0.2226 nM, respectively. Moreover, the nanocomposite also provides a feasible platform for cytotoxicity screening in cancerous cells (Caco-2 cell lines) and non-cancerous cells (L-929 cell lines).