Based on the principle that the nitro-group can quench the fluorescence and can be reduced under hypoxic conditions, several novel nitroheterocyclic compounds without 2-nitroimidazole as potential hypoxic markers were prepared. Although they were synthesized from the same matrix, nitrosubstituted acenaphtho[1,2-b]quinoxaline, these compounds exhibited quite different fluorescence changes when they were differently nitrosubstituted. Their evaluation for imaging tumor hypoxia was carried out in V79 cells in vitro by Fluorescence Microplate Reader. After 3.5 h, the hypoxic-oxic fluorescence differential incubated with A1, A4, and A5 in V79 cells could reach 6, 9, and 11 times differential fluorescence between oxic and hypoxic cells separately, which are suitable for further evaluation as probes for hypoxic cells in tumors in vivo.