Upconversion nanoparticles (UCNPs) have achieved considerable success in protein sensing in vitro. And aptamer is one of the most frequently used biomolecules to modify the nanoparticles for protein assay. However, the complicated process of modifying UCNPs with DNA and the susceptibility of the phosphate groups of DNA backbone to adsorb on the surface of UCNPs have limited their practical applications. To overcome these limitations, a modification-free fluorescent biosensor based on polydopamine-coated upconversion nanoparticles (UCNPs@PDA) is proposed. It consists of UCNPs@PDA and CEA aptamer-functionalized AuNPs (AuNPs-CEA aptamer). The CEA aptamer on AuNPs can be adsorbed onto the surface of UCNPs@PDA due to the interactions of π-π stacking and hydrogen bonding, triggering the process of fluorescence resonance energy transfer (FRET) from UCNPs@PDA to AuNPs-CEA aptamer. In the presence of CEA, the AuNPs-CEA aptamer departs from UCNPs@PDA due to the stronger affinity of CEA with its aptamer. Therefore, the recovery of upconversion fluorescence can sensitively quantify the concentration of CEA. This biosensor provides a linear range from 0.1 to 100 ng/mL for CEA with a LOD of 0.031 ng/mL in an aqueous solution. In spiked human serum samples, the same linear range is acquired with a slightly higher LOD of 0.055 ng/mL, demonstrating the great potential of the biosensor in practical application.