Marine scallops are sessile as adult but have a long planktonic larval phase showing great possibility to migrate in marine realm lacking of obvious barriers. Genetic analysis of scallop embryos/larvae based on molecular markers is very essential to clarify the spatial and temporal gene flow and the unique population and community structure. However, the technical challenges, such as single embryos/larvae isolation and low quantity and poor quality of DNA extracted, make genotyping for a single embryo/larva long preserved in ethanol to be a really difficult task. In this study, we analysed the factors that might affect the DNA quantity and quality for simple sequence repeat-based genotyping for single embryos/larvae. Based on the factors analysed, we developed a LoTEPA buffer-based method, of which the accuracy, stability and reproducibility were evaluated by controlled inter- and intraspecies and self-fertilized scallop families. The genotyping results showed the high success rate of more than 90% in total for embryos/larvae preserved in ethanol for 1–5 years. Furthermore, the successful genotyping for the larvae sampled from a natural habitat well demonstrated the potential use of this method in practical ecological analysis.