Sample barcoding-based multiplex single-cell and single-nucleus sequencing (sc/sn-seq) offers substantial advantages by reducing costs, minimizing batch effects, and identifying artifacts, thereby advancing biological and biomedical research. Despite these benefits, universal sample barcoding has been hindered by challenges such as inhomogeneous expression of tagged biomolecules, limited tagging affinity, and insufficient genetic insertion. To overcome these limitations, we developed Toti-N-Seq, a universal sample multiplex method, by tagging Toti-N-glycan on cell surfaces or nuclear membranes via our engineered streptavidin–Fbs1 GYR variant fusion protein, which could be used not only for sc-seq but also for sn-seq. Instead of targeting lipids or proteins, we focused on targeting the ubiquitous N-glycans found on any species with accessible membranes, which minimizes the exchange between barcoded samples and avoids biased barcoding. Our technology can be broadly applied to multiple species and nearly all eukaryotic cell types, with an overall classification accuracy of 0.969 for sc-seq and of 0.987 for sn-seq. As a demonstration with clinical human peripheral blood mononuclear cells, our Toti-N-Seq achieved rapid one-step sample preparation (<3 min) for easily scaling up while keeping high fidelity of sample ratios, removing artifacts, and detecting rare cell populations (~0.5%). Consequently, we offer a versatile platform suitable for various cell types and applications.