Background: Growing insecticide resistance and changes in biting and resting behavior of malaria vectors threaten efficacy of insecticide treated nets and indoor residual spraying. Larval source management (LSM) is a promising approach that can target mosquitoes irrespective of their behavior as adults. However, the use of traditional monitoring methods for immature stages of Anopheles mosquitoes is a major challenge to LSM due to the variability in their breeding habitats. We evaluate the use of an environmental DNA (eDNA) analysis technique in monitoring Anopheles gambiae sensu lato larvae in experimental aquatic habitats. Methods: eDNA was simultaneously sampled and extracted from different volumes of water, number of larvae, and occupation time. Larval presence was detected using PCR and eDNA concentration in samples from 1 L habitats quantified using an IGS and cyt b TaqMan assays. The limit of detection of the two assays was tested and larval density correlated with eDNA positivity. Results: 74% of replicates in the 50 mL habitats were PCR positive with at least 6h required to get a signal from a single larva (0.02 larvae/mL). All 12 replicates where 1 L of water was used were positive with stronger PCR bands than replicates with the same larval density in 50 mL for 24 h. There was a correlation between larval densities and eDNA detection in both assays: IGS, r = 0.503, p = 0.047; and cyt b,r = 0.558, p = 0.025. There was stochasticity in eDNA detection rates, using both PCR and qPCR across all the dilutions. Conclusion: This study has demonstrated the potential use of eDNA analysis for detection and quantification of An. gambiae s.s. mosquito larvae in aquatic habitats. The stochasticity observed in eDNA detection suggest that this technique is best for monitoring aquatic habitats with many larvae at low densities.