Aqueous synthesis is a green but challenging pathway to produce cadmium-free, bright, and near-infrared (NIR) emitting quantum dots (QDs). Herein, we develop a straightforward aqueous phase approach to prepare Zn-Cu-In-Se QDs with tunable emission from 630 to 800 nm through varying the molar ratio of Zn and Cu. The ultrasmall nanoparticle size remains nearly fixed (~ 2.6 nm) when the QD composition is varied, allowing for the effects of composition on the optical properties of QDs to be decoupled from the impact of particle size. By leveraging the off-stoichiometric effect on photoluminescence (PL), Zn-Cu-In-Se QDs with a Zn:Cu molar ratio of 1.0 show a long PL decay lifetime of 211 ns and an absolute photoluminescence quantum yield (PLQY) of 14.2%. The PLQY is further enhanced by overcoating a ZnS shell, reaching up to 25.8%. Based on the bright, biocompatible, and emission-tunable QDs, nanoprobes with targeting capability for multiple tumor markers are further constructed and employed to simultaneously ascertain targets in the cytoplasm and cell membrane. This study provides a green and effective strategy for achieving bright and biocompatible NIR quantum dots for multiplexed biodetection applications.