AbstractRecent developments in environmental DNA (eDNA) analysis allow more rapid and extensive biomonitoring than traditional capture‐based surveys do. However, detection of eDNA not derived from living organisms may lead to false‐positive inferences of species presence. Such limitations may be overcome by utilizing RNA molecules present in the environment (environmental RNA [eRNA]) because of their physiochemical instability. Nevertheless, the biomonitoring performance of eRNA analysis remains unclarified because of the substantial lack of knowledge regarding basic eRNA properties, such as its persistence and degradation mechanisms. Here, we performed a factorial aquarium experiment to assess the effects of water temperature (10, 20, and 30°C) and pH (4, 7, and 10) conditions on the degradation of zebrafish (Danio rerio) eDNA and eRNA, targeting the mitochondrial cytochrome b (CytB) and nuclear beta‐2‐microglobulin (b2m) genes. A linear mixed‐model analysis showed that the degradation of eRNA was significantly faster than that of eDNA. Higher water temperatures promoted both eDNA and eRNA degradation, and alkaline conditions substantially promoted eRNA but not eDNA degradation. This might be explained by the physicochemical characteristics of DNA and RNA molecules, the membranous structure surrounding them, and their susceptibility to environmental microbial activity. Moreover, the relative concentrations of zebrafish eRNA to eDNA decreased over time, inferring that the ratio of eRNA to eDNA concentrations can be used for estimating the elapsed time since the genomic material was released and the freshness of the target eDNA signal in the field. Nevertheless, given that the confidence intervals of the eDNA and eRNA decay rates tended to overlap for each treatment level, this study indicates that fish eRNA is not always degraded rapidly and is, in fact, more abundant in water than previously expected. This result favors the application of eRNA analysis to indicate living biotic assemblages.