Natural polymer-based sutures have attractive cytocompatibility and degradability in surgical operations. Herein, anionic cellulose nanofibrils (ACNF) and cationic guar gum (CGG) are employed to produce nontoxic CGG/ACNF composite filament with a unique core-shell structure via interfacial polyelectrolyte complexation (IPC) spinning. The comprehensive characterization and application performance of the resultant CGG/ACNF filament as a surgical suture are thoroughly investigated in comparison with silk and PGLA (90% glycolide and 10% l-lactide) sutures in vitro and in vivo, respectively. Results show that the CGG/ACNF filament with the typical core-shell structure and nervation pattern surface exhibits a high orientation index (0.74) and good mechanical properties. The tensile strength and knotting strength of CGG/ACNF suture prepared by twisting CGG/ACNF filaments increase by 69.5%, and CGG/ACNF suture has a similar friction coefficient to silk and PGLA sutures. Moreover, CGG/ACNF suture with antibiosis and cytocompatibility exhibits better growth promotion of cells than silk suture, similar to PGLA suture in vitro. In addition, the stitching experiment of mice with the CGG/ACNF suture further confirms better healing properties and less inflammation in vivo than silk and PGLA sutures do. Hence, the CGG/ACNF suture with a simple preparation method and excellent application properties is promising in surgical operations.