The enhancement of upconversion luminescence (UCL) of rare earth doped upconversion nanoparticles (UCNPs) in aqueous solution is particularly important and urgently required for a broad range of biomedical applications. Herein, an effective approach to achieve highly enhanced UCL from NaYF4:Yb3+,Tm3+ UCNPs in aqueous solution is presented. We demonstrate that UCL of these UCNPs can be enhanced more than 104-fold by using a mesoporous silica low refractive index resonant waveguide grating (low-n RWG) in contact with aqueous solution, which makes it well-suited for biomedical applications. The structure parameters of the low-n RWG are tuned via rigorous coupled-wave analysis simulation to ensure strong local excitation field to form atop the TiO2 surface of the low-n RWG, where UCNPs are deposited. As the low-n RWG is excited by a near-infrared laser at 976 nm to match its guided mode resonance (GMR) condition, UCL emitted from UCNPs is greatly enhanced thanks to the strong interaction between excitation local field and UCNPs. UCL emission of UCNPs can be further enhanced about two to four times when the UCL emission condition (wavelength and angle) matches with the GMR condition. Furthermore, we show that the presence of biotin molecules atop of the low-n RWG can be easily detected through UCL emission generated from streptavidin-functionalized UCNPs with the help of the streptavidin–biotin specific binding. The results indicate that the low-n RWG has high potential for UCL biosensing and bioimaging applications.