Abstract: Line transect surveys are widely used in Neotropical rainforests to estimate the population abundance of medium- and large-sized vertebrates. The use of indices such as encounter rate has been criticized because the probability of animal detection may fluctuate due to the heterogeneity of environmental conditions among sites. In addition, the morphological and behavioral characteristics (biological traits) of species affect their detectability. In this study, we compared the extent to which environmental conditions and species’ biological traits bias abundance estimates in terra firme rainforests in French Guiana. The selected environmental conditions included both physical conditions and forest structure covariates, while the selected biological traits included the morphological and behavioral characteristics of species. We used the distance sampling method to model the detection probability as an explicit function of environmental conditions and biological traits and implemented a model selection process to determine the relative importance of each group of covariates. Biological traits contributed to the variability of animal detectability more than environmental conditions, which had only a marginal effect. Detectability was best for large animals with uniform or disruptive markings that live in groups in the canopy top. Detectability was worst for small, solitary, terrestrial animals with mottled markings. In the terra firme rainforests that represent ~80% of the Amazonia and Guianas regions, our findings support the use of relative indices such as the encounter rate to compare population abundance between sites in species-specific studies. Even though terra firme rainforests may appear similar between regions of Amazonia and the Guianas, comparability must be ensured, especially in forests disturbed by human activity. The detection probability can be used as an indicator of species’ vulnerability to hunting and, thus, to the risk of local extinction. Only a few biological trait covariates are required to correctly estimate the detectability of the majority of medium- and large-sized vertebrates. Thus, a biological trait model could be useful in predicting the detection probabilities of rare, uncommon, or localized species for which few data are available to fit the detection function.