We have designed and investigated a three-band refractive index (RI) sensor in the range of 550–900 nm based on the metal nanoslit array with gain-assisted materials. The underlying mechanism of the three-band and enhanced characteristics of the metal nanoslit array with gain-assisted materials, have also been investigated theoretically and numerically. Three resonant peaks in transmission spectra are deemed to be in different plasmonic resonant modes in the metal nanoslit array, which leads to different responses for the plasmonic sensor. By embedding the structure into the CYTOP with proper gain-assisted materials, the sensing performances can be greatly enhanced due to a dramatic amplification of the extraordinary optical transmission (EOT) resonance by the gain medium. When the gain values reach their corresponding thresholds for the three plasmonic modes, the ultrahigh sensitivities in three bands can be obtained, and especially for the second resonant wavelength (λ₂), the FOM=128.1 and FOM^* = 39100 can be attained at the gain threshold of k =0.011. Due to these unique features, the designing scheme of the proposed gain-assisted nanoslit sensor could provide a powerful approach to optimize the performance of EOT-based sensors and offer an excellent platform for biological sensing.