Cytochrome P450 enzymes (P450s) have gained significant attention due to their remarkable ability to oxidise unactivated C-H bonds with high regio- and stereoselectivity. Their industrial utility is often limited by challenges such as low stability, poor expression, and dependence on elusive redox partners. These issues have driven the search for more robust P450s, especially those that are inherently stable under extreme conditions typical of industrial processes. Self-sufficient P450s in which the P450 heme domain is naturally fused to redox domains in a single polypeptide chain eliminates the need to identify and separately express required redox partners. Furthermore, P450s from thermophilic organisms are more temperature tolerant with fewer stability issues. This study presents a self-sufficient P450, CYP116B305, identified from metagenomically assembled genomes from Innot Hot Springs (71 °C), located in North Queensland, Australia. CYP116B305 was heterologously expressed in Escherichia coli and purified using standard protocols. Investigation of the thermal stability of CYP116B305 revealed a robust heme domain with a 15T50 value of 56.9 ± 0.1 °C, while the reductase domain exhibited slightly lower stability, with a 15T50 value of 52.5 ± 0.5 °C. Further characterisation revealed that CYP116B305 efficiently bound to and hydroxylated 2-hydroxyphenylacetic acid (2-HPA) at the C-5 position, yielding homogentisic acid. The catalytic parameters, including the coupling efficiency and rate of electron transfer from the NADPH cofactor to the P450 heme, were shown to improve at an elevated temperature (45 °C) compared to 25 °C. The combination of the self-sufficiency and improved stability makes CYP116B305 a promising platform for biotechnological applications and biocatalyst engineering. KEY POINTS: • Hot spring metagenomics reveals thermostable P450s of biocatalytic value. • CYP116B305 shows enhanced catalytic activity at elevated temperature (45 °C). • CYP116B305 is a promising platform enzyme for diverse biotechnological use.