Circulating plasma microRNAs (miRNAs) are well established as biomarkers of several diseases in humans and have recently been used as indicators of environmental exposures in fish. However, the role of plasma miRNAs in regulating acute stress responses in fish is largely unknown. Tissue and plasma miRNAs have recently been associated with excreted miRNAs in humans however external miRNAs have never been measured in fish. The objective of this study was to characterize the plasma miRNA profile in response to acute stress in rainbow trout (Oncorhynchus mykiss), as well as miRNA profiles in novel external samples, (fish epidermal mucus and the surrounding water). RNA was extracted and sequenced from plasma, mucus, and water collected from rainbow trout and their surrounding environment prior to and one-hour following a three-minute air exposure, a known inducer of an acute stress response in fish. Following small RNA-Seq and pathway analysis, we identified differentially expressed plasma miRNAs that targeted biosynthetic, degradation, and metabolic pathways. We successfully isolated miRNA from trout mucus and the surrounding water and detected differences in miRNA expression one-hour post air stress. The altered miRNA profiles in mucus and water were unique to the altered plasma miRNA profile, indicating that the plasma miRNA response was not associated with or immediately reflected in external samples. This research expands our understanding of the role of plasma miRNA in the acute stress response of fish and is the first study to report on the successful isolation and profiling of miRNA from fish mucus and water samples. Measurements of miRNA from plasma, mucus, and water can be further studied and have the potential to be applied in environmental monitoring as non-lethal indicators of acute stress in fish. Overall design: Six blood plasma samples, six epidermal mucus samples, and six water samples were analyzed. Each of these had three experimental replicates for the control treatment and three experimental replicates for the stressed treatment (18 samples total).