Although multifunctional upconversion imaging probes have recently attracted considerable interest in biomedical research, there are currently few methods for stabilizing these luminescent nanoprobes with oligonucleotides in biological systems. Herein, a method to robustly disperse upconversion nanoprobes in physiological buffers based on rational design and synthesis of nanoconjugates comprising hairpin-DNA-modified gold nanoparticles is presented. This approach imparts the upconversion nanoprobes with excellent biocompatibility and circumvents the problem of particle agglomeration. By combining single-band anti-Stokes near-infrared emission and the photothermal effect mediated by the coupling of gold to upconversion nanoparticles, a simple, versatile nanoparticulate system for simultaneous deep-tissue imaging and drug molecule release in vivo is demonstrated. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim