The environmental DNA (eDNA) technique is expected to become a powerful tool for estimating the distribution and biomass of aquatic vertebrates. However, basic information on eDNA release rates is lacking. Here, we examined the effect of fish size on eDNA release velocity using bluegill sunfish. The eDNA concentration reached equilibrium 2 days after the fish introduction to the containers, and the eDNA release velocity was 3–4 times higher in the adult (30–75 g) than in the juvenile group (0.5–2.0 g). However, the eDNA release velocity per fish body weight was slightly higher in the juvenile than the adult group, probably because of the size effect on metabolism. The positive relationship between fish size and eDNA release suggested the possibility of biomass estimation using eDNA techniques. However, quantitative eDNA data should be carefully interpreted to avoid overestimating the population biomass dominated by juveniles, because the size composition of the focal population is often variable and unseen in the field. Decomposition rates of eDNA, calculated by curve fitting of the time-dependent change in eDNA concentrations after fish elimination, were 5–16% per hour. This suggests that quantitative eDNA data should be corrected using a decomposition curve attained in the target field.