We report the development of an azoreductase-responsive prodrug AP-N═N-Cy in which the precursor compound AP, a readily available podophyllotoxin derivative, is linked with a NIR fluorophore (Cy) via a multifunctional azobenzene group. This type of azo-based prodrug can serve as not only an azoreductase-responsive NIR probe to real-time tracking of the drug delivery process but also a delivery platform for an anticancer compound (AdP). We have shown that cleavage of the multifunctional azobenzene group in AP-N═N-Cy only occurred in the presence of azoreductase, which specifically secretes in the colon, resulting in direct release of AdP through an in situ modification of a phenylamino group on the precursor AP. Moreover, introduction of the azobenzene group endows the prodrug with an unique fluorescence "off-on" property and served as a switch to "turn on" the fluorescence of Cy as consequence of a self-elimination reaction with breakage of an azo bond. Such a prodrug can be administered orally and exhibit high stability and low toxicity before arriving at the colon. In view of the synchronism of drug release and the fluorescence turn-on process, the fluorescence imaging method was utilized to precisely trace drug delivery in vitro, ex vivo, and in vivo. Distinguishingly, the biodistribution of AdP and Cy in various tissues was further precisely mapped at the molecular level using imaging mass spectrometry. To the best of our knowledge, this is the first time that the in vivo real-time precise tracking of the colon-specific drug release and biodistribution was reported via a multimodal imaging method.