This study describes a novel ratiometric fluorescent sensor based on chemical etching of gold nanocluster (GNCs) for label-free, separation-free determination of tris(2-carboxyethyl)phosphine (TCEP). TCEP was discovered to exhibit unusual chemical behavior toward fluorescent gold nanoclusters: it quenched the red fluorescent emission of the bovine serum album (BSA)-protected GNCs (GNCs@BSA) and simultaneously restored the blue fluorescent emission of the dityrosine (diTyr) residues of the BSA ligand. The TCEP-induced quenching of the fluorescent GNCs@BSA was investigated with the UV-vis adsorption spectrum, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), inductively coupled plasma-mass spectrometry (ICP-MS) and X-ray photoelectron spectroscopy (XPS), revealing the chemical etching of the gold(0) core of the GNCs@BSA by TCEP. Furthermore, the ratio of the blue fluorescence intensity of the diTyr to the red fluorescence intensity of the GNCs@BSA was found to be dependent on TCEP concentration and showed a linear relationship in the TCEP concentration range of 500 nM to 50, 000 nM (R2 = 0.9943) with a limit of detection (LOD) of 130 nM, achieving the higher sensitivity over previous reports. This ratiometric sensor also showed superior selectivity for TCEP over certain common interferences including glutathione, 20 kinds of natural amino acids, and the oxidized form of TCEP. With the developed ratiometric method, the deproteinized human serum samples spiked with TCEP were analyzed with satisfactory results. In addition, it is worth noting that compared with conventional ratiometric fluorescent sensors, the ratiometric sensor developed in this study does not require external fluorophores, avoiding the additional derivation procedures.