Cytosensing of biological transition metals is paramount important for cancer research. One-pot synthesis of the nanocomposite of carbon dots (CDs) and gold nanoparticles (AuNPs) is reported and has been applied for metal cytosensing. The nanocomposite (GCDs) is synthesized using citric acid (CA) and L-cysteine as precursors in the presence of chloroauric acid. The synthesis procedure exhibits many advantages including simple one step, green approach (solvent free method) and requires cheap chemical precursors. The current procedure produces uniform carbon dots (<3 nm) and assist the formation of AuNPs without using extra reducing agent. GCDs nanoparticles have UV absorbance matching with the wavelength of N2 laser (337 nm). The synergistic effect of the large surface area of GCDs and the maximum absorbance at 337 nm offers an effective and promising application for surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) of biological metals (Fe2+, Fe3+ and Cu2+ ions) for cancer cells. SELDI-MS using GCDs offers a sensitive method, shows selective detection, and provides simultaneous cytosensing of biological metals (Fe2+, Fe3+ and Cu2+) of cancer cells. The metals are detected after complexation with mefenamic acid (MFA) which provides multi-functions (chelating agent, internal calibrant and co-matrix) for LDI-MS