AbstractDuring the outmigration of Pacific Salmon, the early marine phase is a critical period when high mortality can occur. Traditional sampling and monitoring of juvenile salmon migration can be limited by logistically intensive gear requirements, accessibility, and cost. Improved understanding of the early marine phase, for example, migration duration and habitat use, requires innovative techniques that can improve the spatial and temporal coverage of monitoring. Environmental DNA (eDNA) is genetic fragments present in the environment that can be used as a proxy for organism presence and can be effectively and efficiently collected through water samples. Estimating fish abundance or biomass from eDNA concentration data would provide a valuable fisheries tool but remains challenging to calibrate. To quantify the relationship between eDNA abundance and fish biomass, we used a controlled mesocosm experiment, in which eDNA samples were collected from 15 aquaria (340 L) with varying densities of juvenile Chinook salmon per tank (0, 5, 10, 20, and 30). The concentration of eDNA was obtained by qPCR scaled with fish biomass (ANOVA, p < 0.05). However, we also observed that variability of eDNA concentrations among replicates of the same treatment positively scaled biomass (ANOVA, p < 0.05). Therefore, higher biomasses of fish can yield more challenging data to interpret. This study lays important groundwork for the application of eDNA for monitoring juvenile salmonids yet highlights caveats for the applicability of eDNA as a stand‐alone method to assess biomass in a field setting.