The use of Next-Generation Sequencing (NGS) in environmental DNA (eDNA) research offers increased sensitivity in detecting rare species within vast marine ecosystems. However, conventional NGS methods often fail to accurately capture low-frequency variants, posing risks of imprecise detections. Inspired by clinical research, innovations like SiMSen-Seq (simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection using sequencing) have emerged. SiMSen-Seq utilizes molecular barcoding to track individual DNA templates, correcting polymerase-induced errors and quantification biases. In this study, we adapted the SiMSen-Seq protocol for eDNA analysis, targeting 18S rRNA metabarcoding in seawater samples to enhance marine biodiversity assessment, particularly for identifying rare non-indigenous species (NIS). Comparative analysis against an Illumina MiSeq protocol (Illumina—DADA2) and the SiMSen-Seq protocol with DADA2 bioinformatics (SiMSen-Seq—DADA2) revealed that SiMSen-Seq detected a higher number (1495) of molecular Operational Taxonomic Units (mOTUs) compared to the other two pipelines (Illumina—DADA2: 585 and SiMSen-Seq—DADA2: 578). In terms of genera, SiMSen-Seq detected the lowest number of genera, Illumina—DADA2 the highest number, and 45% of genera were detected across protocols. While all three protocols identified the predominant NIS, they differed in detecting rare taxa: SiMSen-Seq uniquely detected Arenigobius bifrenatus and Pseudopolydora paucibranchiata but did not detect some others (e.g., Asterias amurensis/A. forbesi). These findings suggest that for some species (especially Stylea plicata and Ciona savignyi), SiMSen-Seq could potentially be useful to detect intraspecific variability for population genetic studies, with uses in more precise ecological monitoring and effective marine conservation.