Abiotic stress is a major bottleneck for crop productivity. To counter abiotic stresses, plants have developed several strategies, and the accumulation of polyamine (PA) serving as an osmolyte is one of them. The cellular pool of PAs is primarily regulated by polyamine oxidases (PAOs) either by terminal catabolism or by back conversion (BC) of polyamines. The role of PAO in abiotic stress tolerance has also been reported. Polyamine oxidases are primarily localized in the cytosol, cell wall, and apoplastic regions; however, lately, their peroxisomal localization has also been demonstrated. In this study, we reported the detection of polyamine oxidase isoform 3 from Oryza sativa (OsPAO3) in peroxisome as early as 12 h post-transformation under in vitro conditions using fluorescent microscopy. The gene was also found to be significantly upregulated by salinity, dehydration, cold, and heat stress. We have also demonstrated that the expression of OsPAO3 gene was mediated by a second messenger, calcium. The upregulation of OsPAO3 by salinity, dehydration, cold, and heat stresses suggests that it could be a suitable candidate for providing tolerance to plants against abiotic stress combination or stress matrix, which is a common feature in agricultural field conditions. Furthermore, the data provided here would be valuable in understanding the abiotic stress-mediated signal transduction network of PAOs.