Large marine predators feeding on fish caught on fishing gear, a behaviour termed as depredation, can incur socio-economic costs for fisheries and have serious implications on the depredating species, fish stocks and associated ecosystems. The quantity of depredated fish as well as the quantity of depredated fish relative to the retained catch dictates the severity of these impacts, yet these depredated quantities are often unknown due to the complexity of assessing depredation. This is the case for many demersal longline fisheries experiencing depredation by killer whales (Orcinus orca), which remove entire fish from hooks when interacting with the fishing gear. In the present study, we used Generalized Linear Models fitted to the Catch per Unit Effort (CPUE) to quantify killer whale depredation in the blue-eye trevalla (Hyperoglyphe antarctica) commercial longline fishery of south-east Australia. Our results showed that during days when killer whales were present around fishing vessels (7.3% of total fishing days), blue-eye trevalla CPUE decreased by 45.6%. Killer whales were estimated to have removed a total of 63.9 t (95% CI: 45.3–82.7 t) of blue-eye trevalla from the hooks between 2006 and 2017, with a mean removal of 5.3 t (3.8–6.9 t) per year. The mean rate of killer whale depredation in south-east Australia was estimated to be 5.2% (4.0–6.4%) for the entire study period. We estimated that the current depredation rate equates to an additional 27 fishing days per year across the fishery. This figure grows to 354 additional fishing days if depredation was assumed to occur during every fishing operation. Considering that killer whale observations are voluntarily reported in logbooks, this likely underestimates the depredation in our study. Together, these findings suggest that the impacts of killer whale depredation in the blue-eye trevalla fishery of south-east Australia are not negligible given the small scale of the fishery and the historical decline of its catches in this region. We recommend estimates provided in this study to be used as baselines for the future assessment and management of the blue-eye trevalla stocks and for assessing the socio-economic and ecological implications of this issue. In addition, regardless of the limitations associated with assessing depredation, our study also highlights the importance of cross-sectoral and interdisciplinary collaborations between scientists, fishers and fishery managers for effectively improving the management of commercially important fisheries globally.