Upconversion nanoparticles (UCNPs) are new optical probes for biological applications. For specific biomolecular recognition to be realized for diagnosis and imaging, the key lies in developing a stable and easy-to-use bioconjugation method for antibody modification. Current methods are not yet satisfactory regarding conjugation time, stability, and binding efficiency. Here, we report a facile and high-yield approach based on a bispecific antibody (BsAb) free of chemical reaction steps. One end of the BsAb is designed to recognize methoxy polyethylene glycol-coated UCNPs, and the other end of the BsAb is designed to recognize the cancer antigen biomarker. Through simple vortexing, BsAb-UCNP nanoprobes form within 30 min and show higher (up to 54%) association to the target than that of the traditional UCNP nanoprobes in the ELISA-like assay. We further demonstrate its successful binding to the cancer cells with high efficiency and specificity for background-free fluorescence imaging under near-infrared excitation. This method suggests a general approach broadly suitable for functionalizing a range of nanoparticles to specifically target biomolecules.