Micron-scale patterning of colloidal quantum dots (QDs) is extremely important for the fabrication of high-performance Quantum dot Light-Emitting Diode (QLED) displays, biosensing, and super-resolution imaging. Thus, several nondestructive methods have been recently proposed, such as spatial self-organization. However, none of them can be useful for biofunctionalization or nanoimaging. To address this limitation, we propose a method to create micropatterns of QDs of any shape and size. UV photolithography assisted by a digital micromirror device (DMD) and silanization allow creating an adhesive layer, on which QDs micropatterns can be assembled with a 2 μm resolution. The patterns are composed of a monolayer of CdSe/CdS/CdZnS/ZnS core/multishell QDs (7 ± 1 nm in diameter, emitting at 590 nm) with a high surface density (typically 4000 QDs/μm2). We also demonstrate that it is possible to reversibly bind any kind of His-Tagged proteins on the QDs surface. This is highlighted by measuring FRET (Förster Resonance Energy Transfer) with a dedicated polymer exhibiting on one end Alexa Fluor 647 (AF647) and on the other end eight imidazole cycles, allowing chelation on the quantum dots’ surface. Therefore, this patterning protocol provides a path to combine nanoimaging with cell patterning through a relevant biofunctionalization.