Bioimaging of fluorescent probes provides a fantastic tool to visualize different cellular responses from various cells and to monitor target/probe interaction in cells. Previously, we introduced a small molecule named 3,6-bis(1-methyl-4- vinylpyridinium)carbazole diiodide (BMVC) as a fluorescent marker to light up live cancer cells for cancer diagnosis. Here we used time-gated fluorescence lifetime imaging microscopy (FLIM) of a BMVC isomer, 3,6-bis(1-methyl-2- vinylpyridinium)carbazole diiodide (o-BMVC) as a fluorescent probe to distinguish fixed cancer cells for cancer diagnosis. The time-gated FLIM results showed many more number of o-BMVC foci, characterized by the longer fluorescent decay time of o-BMVC (≥2.4 ns), in fixed cancer cells (HeLa, H1299, CL1-0, MCF-7, MCF-7/ADR, and SAS) than in fixed normal cells (MRC-5, IMR-90, and BJ). Further study of tissue biopsy, a total of 50 head and neck cancer (HNC) samples obtained during surgery and 20 normal oral samples collected from healthy volunteers showed that o-BMVC foci are hardly detectable in the normal oral epithelial cells. The average numbers of o-BMVC foci in tumors and in normal oral epithelial cells are 28.3 and 2.2, respectively. The receiver operating characteristic curve analysis showed the area under curve was 0.992, indicating that this method provides a very high accuracy for clinical detection of HNC cancers. We propose that the combination of BMVC test for live cells and o-BMVC test for fixed cells could provide a powerful tool for screening of human cancers.