The response surface methodology was used to evaluate the influence of salt content (5–20%), fermentation temperature (25–50 °C), and fermentation time (20–80 days) on the chemical characteristics and histamine level of Iranian fish sauce (Mahyaveh) prepared from Anchovy fish. The results showed that salt had a significant quadratic effect on pH, total nitrogen, salt, and histamine content. The total nitrogen, pH, and histamine of fish sauce decreased with increasing salt concentration. Salt content had a significant linear effect on amino nitrogen. The amount of amino nitrogen decreased with increasing salt. Time had a positive quadratic effect on histamine and total nitrogen levels in fish sauce, whereas it had a positive linear effect on amino nitrogen level. In contrast, temperature showed a significant effect on the total nitrogen, histamine, salt, and pH of fish sauce (p < 0.05). As the temperature increased, total histamine decreased whereas nitrogen concentration increased. The predicted optimal conditions as determined by Response Surface Methodology were salt content of 12.5%, fermentation temperature of 39.15 °C, and fermentation time of 39.79 days. Therefore, high quality Iranian fish sauce can be produced with low salt and histamine content as a potential functional food and ingredient.

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