A new fluorescent sensor is introduced to analyze nucleoside analogue, trifluridine and tipiracil in tablets and biological fluids. The synthesized fluorophore exhibits good fluorescence at 446 nm after excitation at 257 nm. The interaction between the studied drugs and the reagent was a quenching effect. Different experimental parameters and the mechanism of quenching were discussed. The present method was utilized to analyze trifluridine and tipiracil raw materials and tablets over the concentration range of 20–1000 ng/mL and spiked biological fluids over the range of 30–1000 ng/mL. The method is selective, specific, and possesses good accuracy and high precision. The method is highly sensitive, with detection limits of 5.8 and 6.0 ng/mL for trifluridine and tipiracil, respectively, and quantitation limits of 17.7 and 18.1 ng/mL for trifluridine and tipiracil, respectively. In vivo analysis of trifluridine was achieved selectively and the mean pharmacokinetic parameters were studied.