A two-photon fluorescent dye taking isoquinolinone as the parent is provided with a structure presented in formula I, wherein R1 is selected from -H, -CN, -OCH3, -COOH, -OH, -NO2, -NH2, -NHCH3, -N(CH3)2, -OCOCH3 and -SH; R2 is selected from -N-, -C-, -S- and -O-; and R3 is selected from -NHCH2OH, -NH(CH2)2OH, -NH(CH2)3OH, -NH(CH2)4OH,-N(CH3)CH2OH, - N(CH3)(CH2)2OH, -N(CH3)(CH2)3OH and -N(CH3)(CH2)4OH. The fluorescent dye is efficient in two-photon absorptive capacity, low in photo-induced toxicity and photobleaching, and strong in specificity for ribonucleic acid (RNA) in cells; and can perform specificity marking on the RHA in living cells and fixed cells simultaneously. The formula I is shown in the description.