The invention provides a multifunctional near infrared fluorescent probe. The chemical formula of the multifunctional near infrared fluorescent probe is as shown in formula (I), wherein X<1> is -S- or -Se-, R' is C1-12 alkylene, X<2> is -C(CH3)2-, -O- or -Se-, each of R1 and R2 is independently selected from H atoms, C1-18 alkyl or groups as shown in SO3R9, R9 is C1-18 alkyl or benzyl, each R3 and R4 is independently selected from C1-18 alkyl or benzyl, each of R5, R6, R7 and R8 is independently selected from C1-18 alkyl or C1-18 alkyl containing at least one of F, Cl, Br, I, O and S, and each of Y1 and Y2 is independently selected from F, Cl, Br or I. The multifunctional near infrared fluorescent probe can simultaneously detect pH and mercaptan, is a double-response ratio type near infrared fluorescent probe and can be used for the photodynamic therapy of tumors. The invention further provides a preparation method and application of the probe.