In methylotrophic yeasts such as Komagataella phaffii (syn Pichia pastoris), the initial step of methanol metabolism by alcohol oxidase (Aox) generates hydrogen peroxide (H2O2) as a potentially toxic byproduct. Introduction of the ratiometric, genetically encoded fluorescent H2O2 biosensor HyPer7 in combination with cultivation in a microbioreactor allowed for the first time to in vivo determine H2O2 dynamics upon methanol utilization (MUT). In line monitoring of H2O2 during growth on glucose or methanol revealed a general increase in biosensor oxidation on methanol, with significant oxidation peaks shortly after methanol addition. HyPer7 also detected low endogenous H2O2 levels occurring during respiratory growth in K. phaffii and its signal responded to both external oxidants and reductants. In strains with different MUT phenotypes (K. phaffii deleted for aox1 and/or aox2), HyPer7 demonstrated that H2O2 production is mainly due to Aox1 activity, and explained why strains possessing only Aox2 (MutS) have superior growth and production capacities compared to the wild-type. In conclusion, we present the first application of an H2O2 biosensor in K. phaffii, offering new insights into methanol metabolism and oxidative stress. The findings hold promise for optimizing yeast cell factories and developing more sustainable production processes with reduced oxidative stress in the future.