Although upconversion nanoparticles (UCNPs) have drawn increasing attention for their unique photophysical characteristics, they suffer from a bottleneck of low luminescence efficiency due to the poor absorption coefficient of Ln 3+ . Dye sensitization has provided thousands-fold enhancement of upconversion luminescence (UCL) in organic solvents because of the remarkably improved light absorption ability, but the sensitization of UCL in aqueous phase is only less than 20 folds by far, with unknown restrictive factors. Herein, the aggregation-caused quenching (ACQ) of dyes is revealed as the most important reason limiting dye sensitization in aqueous phase, and the problem is circumvented through delicately modulating the physical properties of dyes and their assembly manner with UCNPs. By further alleviating energy back transfer (EBT) from Ln 3+ to dyes, more than 600-fold enhancement of UCL is achieved in aqueous phase. The as-obtained dyes modified UCNPs show good biocompatibility and high signal contrast when applied for deep in vivo imaging.suitable for biological applications including bioimaging, biosensing, drug delivery, phototherapy, and so on. [3] However, due to the parity-forbidden nature of the 4f-4f electronic transitions of lanthanide ions, UCNPs suffer from poor light absorption [4] and accordingly extremely low luminescence efficiency and faint brightness, which impairs their performance in practical applications.In a pioneering work of 2012, Hummelen et al. put forward a dye-sensitization strategy to improve UCL intensity in organic solvent. Different from direct absorption of the excitation light by lanthanide ions, heptamethine cyanine with a much larger absorption cross section was employed as an antenna to absorb the energy of photons and transferred its excited-state energy to Ln 3+ ions through a resonance energy transfer pathway. [5] Owing to the dramatically enhanced light absorption ability, thousands-fold enhancement of UCL in organic phase has been achieved through the dye-sensitization process. [6] Considering the requirements of biological applications, efforts have also been devoted to the sensitization of UCL in aqueous phase. [7] Unfortunately, the sensitization efficiency in aqueous phase is badly restricted. In the past few years, although various structures and composition of UCNPs have been tried, this embarrassing situation changes very slowly. [8] Up to now, the highest enhancement factor of dye sensitization of UCL reported in aqueous phase was only 17-fold. [7a] This means that in a biological aqueous environment, the dye-sensitization strategy is not yet able to provide a solution to the bottleneck of UCL brightness. More importantly, the key factors restricting dye sensitization in aqueous phase are unclear so far.In this work, by delicately modulating the properties of dyes as well as their assembly manner with UCNPs, we discovered the factors that decide the sensitization efficiency. In previous dye-sensitized UCL systems in aqueous phase, dyes were assembled wi...