Here, we propose a zwitterionic near-infrared (NIR) fluorophore-tryptophan (Trp) conjugate with a cleavable linker as a minimal-sized versatile platform (MP) for the preparation of peptide ligand-based off-on type molecular probes. The zwitterionic NIR fluorophore in MP undergoes fluorescence quenching via a photoinduced electron transfer mechanism when in close proximity to tryptophan, and nonspecific binding with serum proteins is minimized by the zwitterionicity of the fluorophore. The linker can be cleaved inside cancer cells in response to tumor-associated stimuli. As a proof-of-concept experiment, ATTO655 was covalently linked with Trp via a diarginine linker to form an MP. A cyclic peptide consisting of Arg-Gly-Asp-d-Phe-Lys (cRGD) was used as a cancer-targeting ligand and was conjugated to the MP to form cRGD-MP. The NIR fluorescence of cRGD-MP could be selectively turned on inside the target cancer cells, thereby enabling specific fluorescence imaging of integrin αvβ3-overexpressing cancer cells in vitro and in vivo.