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Functionalized nanoparticles (NPs) are the foundation of diverse applications. Especially, in many biosensing applications, concentrating suspended NPs onto a surface without deteriorating their biofunction is usually an inevitable step to improve detection limit, which remains to be a great challenge. In this work, biocompatible deposition of functionalized NPs to optically transparent surfaces is demonstrated using shrinking bubbles. Leveraging the shrinking phase of bubble mitigates the biomolecule degradation problems encountered in traditional photothermal deposition techniques. The deposited NPs are closely packed, and the functional molecules are able to survive the process as verified by their strong fluorescence signals. Using high‐speed videography, it is revealed that the contracting contact line of the shrinking bubble forces the NPs captured by the contact line to a highly concentrated island. Such shrinking surface bubble deposition (SSBD) is low temperature in nature as no heat is added during the process. Using a hairpin DNA‐functionalized gold NP suspension as a model system, SSBD is shown to enable much stronger fluorescence signal compared to the optical‐pressure deposition and the conventional thermal bubble contact line deposition. The demonstrated SSBD technique capable of directly depositing functionalized NPs may significantly simplify biosensor fabrication and thus benefit a wide range of relevant applications.