PAiRFP1 is one Near-infrared fluorescent protein modified from Agp2, one bacteriophytochrome from Agrobacterium tumefaciens, using protein engineering strategy. Being different from other bacteriophytochrome-based fluorescent protein, PAiRFP1 exhibited specific photoactivatable behavior. This property favored the enhancement of signal-to-noise ratio when PAiRFP1 was used as fluorescence probe in vivo imaging. Little useful information has been available concerning what factors would affect the photoactivation behavior of PAiRFP1. We herein investigated the effect of protein concentration, pH, metal ion and redox condition on the photoactivation of PAiRFP1. It was found that (1) there is no linear relationship between the maximum photoactivation efficiency and the protein concentration. (2) The maximum photoactivation efficiency increased from 36.8% to 52.0% and 60.8% upon increasing pH from 6.5 to 7.8 and 9.0. (3) The metal ions exerted no significant effect on the maximum photoactivation efficiency of PAiRFP1. Similar phenomenon was observed following the treatment of hydrogen peroxide or dithiothreitol. Apart from these factors, one mutant named V276G was constructed, where the valine residue at site 276 was mutated to glycine residue. This displacement resulted in one decrease of the maximum photoactivation efficiency from ~50.0% to 19.4%. Additionally, when DTT was used to the V276G mutant, the maximum photoactivation efficiency was observed to increase from 19.4% to 27.1%. In this paper, we also studied the changes of photoactivation rate induced by these factors. These results will provide useful information and guidance for the application of PAiRFP1 into in-vivo imaging.