The Methylene Blue loaded Polyacrylamide Nanoparticles (MB-PAA NPs) are used for oxygen sensing and Photodynamic therapy (PDT), a promising therapeutic modality employed for various tumors, with distinct advantages of delivery of biomedical agents and protection from other bio-molecules overcoming inherent limitations of molecular dyes. Lifetime-resolved photoacoustic spectroscopy using quenched-phosphorescence method is applied with MB-PAA NPs so as to sense oxygen, while the same light source is used for PDT. The dye is excited by absorbing 650 nm wavelength light from a pump laser to reach triplet state. The probe laser at 810 nm wavelength is used to excite the first triplet state at certain delayed time to measure the dye lifetime which indicates oxygen concentration. The 9L cells (106 cells/ml) incubated with MB-PAA NP solution are used for monitoring oxygen level change during PDT in situ test. The oxygen level and PDT efficacy are confirmed with a commercial oximeter, and fluorescence microscope imaging and flow cytometry results. This technique with the MB-PAA NPs allowed us to demonstrate a potential non-invasive theragnostic operation, by monitoring oxygen depletion during PDT in situ, without the addition of secondary probes. Here, we demonstrate this theragnostic operation, in vitro, performing PDT while monitoring oxygen depletion. We also show the correlation between O2 depletion and cell death.