[Background] Small non-coding RNAs (sRNAs) play important roles in genetic variation, growth, reproduction, and bacterial pathogenicity, but few studies have been conducted on the opportunistic pathogen of Vibrio alginolyticus. We have identified an sRNA, srvg23535, of V. alginolyticus ZJ-T by RNA-seq and Northern Blot. [Objective] We studied the effect of srvg23535 on the biological characteristics of V. alginolyticus. [Methods] The srvg23535 knock-out mutant was constructed by homologous recombination. The function of srvg23535 was studied with comparing the colony morphology, motility, extracellular protease secretion, pressure sensing of H2O2 and Cu2+, iron utilization, antibiotic resistance, and metabolism between the wild type and the mutant strains. [Results] The biological characteristics analysis of srvg23535 knock-out mutant showed that the absence of srvg23535 had no significant effect on the colony morphology, motility, extracellular protease secretion, pressure sensing of H2O2 and Cu2+, iron utilization, antibiotic resistance and the metabolism of most of the tested carbon and nitrogen sources in V. alginolyticus. However, the mutant showed a weaker metabolism of D-trehalose, a stronger metabolism of pectin and alanine-glutamine (Ala-Gln), and accessibility to the utilization of alanine-aspartic acid (Ala-Asp) as the sole nitrogen source. [Conclusion] The sRNA srvg23535 is involved in the metabolism regulation of D-trehalose, pectin, Ala-Gln and Ala-Asp with laying the foundation for the target genes screening of srvg23535 and further elucidating the regulation mechanism of srvg23535 to its target genes.