In vivo photoacoustic (PA) imaging with high spatial resolution and depth penetration has remained a challenge due to the high background signal from chromophores in biological tissue. In this study, we introduce a novel imaging technique called triplet differential (TD) imaging which allows for the substantial reduction of background signals from tissue. TD imaging uses the ability of molecules to enter a triplet state via intersystem crossing from an excited singlet state. Molecules in the triplet state exhibit a spectral shift in their optical absorption spectra, creating two separate absorption peaks for each molecule’s singlet and triplet states. Since the PA signal is proportional to optical absorption, a differential signal can be obtained by comparing the PA signal for molecules raised to the triplet state to those in the singlet state. We worked with methylene blue conjugated polyacrylamide nanoparticles with a polyethylene glycol dimethacrylate cross-linker (MBNP) which has a singlet peak absorption at 660nm and triplet peak absorption at 840nm. Since only certain molecules such as methylene blue can enter the triplet state efficiently, the difference in the PA signal before and after excitation of the MBNP to the triplet state is largely independent of the background noise and mainly contributed by the MBNP in the triplet state. Preliminary results have shown that up to an 8-fold increase in the PA signal of the MBNP in the triplet state can be achieved.