A biopesticide from marine-derived amino-oligosaccharides (AO) are extensively utilized in China, yet there is a lack of scientific literature on their potential ecological risks when transmitted through the food chain to Neoseiulus barkeri, a natural predator of Frankliniella occidentalis. In this study, we conducted a comprehensive investigation into the food chain effects of AO on both F. occidentalis and N. barkeri. Our findings indicate that AO-treated cucumber leaves facilitated the population growth of F. occidentalis but indirectly suppressed the fecundity of N. barkeri which fed on the first instar nymphs of F. occidentalis. Besides, F. occidentalis that ingested AO-treated cucumber leaves exhibited elevated levels of total protein and defense enzymes, including catalase (CAT) and peroxidase (POD), whereas the detoxification enzymes activity, such as carboxylesterase (CarE) and cytochrome P450 monooxygenase (P450), was diminished. Conversely, N. barkeri exhibited decreased levels of the defense enzymes superoxide dismutase (SOD) and POD, coupled with an elevated CarE activity, because of AO food chain transmission. In conclusion, the food chain effects of AO on non-target organisms F. occidentalis and N. barkeri may involve the modulation of defense and detoxification enzyme activities, leading to varying fitness costs. These findings provide critical insights for agricultural pest management strategies, highlighting the necessity of evaluating the effects of AO on non-target organisms within ecosystems, especially beneficial insects, during its application.