Peptide hydrogels were believed to be potential biomaterials with wide application in the biomedical field because of their good biocompatibility, injectability, and three‐dimensional (3D) printability. Most of the previously reported polypeptide hydrogels are composed of L‐peptides, while the hydrogels formed by self‐assembly of D‐peptides are rarely reported. Herein, we reported a peptide hydrogel constructed by D‐J‐1, which was the all‐D‐enantiomer of antimicrobial peptide Jelleine‐1. Field emission scanning electron microscope (FE‐SEM) and rheologic study were performed to characterize the hydrogel. Antimicrobial, hemostatic, and anti‐adhesion studies were carried out to evaluate its biofunction. Our results showed that D‐J‐1 hydrogel was formed by self‐assembly and cross‐linking driven by hydrogen bonding, hydrophobic interaction, and π‐π stacking force of aromatic ring in the structure of D‐J‐1. It exhibited promising antimicrobial activity, hemostatic activity, and anti‐adhesion efficiency in a rat sidewall defect‐cecum abrasion model. In addition, it also exhibited good biocompatibility. Notably, D‐J‐1 hydrogel showed improved in vitro and in vivo stability when compared with its L‐enantiomer J‐1 hydrogel. Therefore, the present study would provide new insight into the application of D‐peptide hydrogel, and provide a new peptide hydrogel with antibacterial, hemostatic, and anti‐adhesion efficacy for clinical use.This article is protected by copyright. All rights reserved