To increase the added value of tuna dark muscle by-product by altering its inclusion in fish sauce, the by-product was hydrolyzed as a raw material and then fermented with soy-wheat koji and salt-tolerant microorganisms. Yellowfin tuna (Thunnus albacares) contains 64.34% moisture, 16.89% protein, 12.62% fat, and 0.92% ash. In this study, 3 volumes (v/w) of distilled water were added to tuna dark muscle through autolysis or enzymatic hydrolysis with the protease (alcalase and flavourzyme), and then hydrolyzed for 4 or 6 hours, respectively. The enzymatic hydrolysis group had much higher peptide levels than the autolysis and control groups did; the alcalase 2 and flavourzyme 4 (A2F4) hydrolysis group demonstrated the greatest increase. The volatile basic nitrogen (VBN), trimethylamine (TMA), and hydrolysis ratio of autolysis or enzymatic hydrolysis are higher than the control group, and A2F4 was the highest. The VBN levels in all the groups were lower than the aquatic product quality limit standard of 25 mg/100 g. According to these results, the A2F4 hydrolysate produced using two-stage complex enzymes can greatly improve taste and functional components such as amino acids and peptides. Thus, this hydrolysate was selected as a raw material for the further fermentation of fish sauce. The salinity of the hydrolysate from the dark muscle was adjusted to 15% and 30%, the hydrolysate was inoculated with 10% soy-wheat koji, and then salt-tolerant microorganisms were added for fermentation at 35 °C for 90 days. The results showed that with prolonged brewing, the L values of the four fish sauces decreased, but the a and b values increased. The pH value slowly declined, and no significant change was observed in salt content. Furthermore, the VBN and TMA values increased with fermentation time, while the soluble solid content increased rapidly until Day 15 and then increased slowly until Day 75. The total nitrogen and amino nitrogen contents increased gradually until Day 60 and then decreased slowly thereafter. The total nitrogen and amino nitrogen of the 15% NaCl product met the fish sauce standards of the Codex Alimentarius Commission (CAC). Overall, the 15% NaCl fish sauce contained a higher amount of total nitrogen and amino nitrogen than did the 30% NaCl product. The histamine contents of the four products were lower than the 40 mg/100 g CAC standard. The addition of salt-tolerant microorganisms during fermentation showed no significant effect on the quality of the fish sauce products. The results revealed that inoculation with soy-wheat koji is a feasible method for making fish sauce from tuna dark muscle by-product.