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In recent years, there has been an increased motivation to reduce meat consumption globally due to environmental and health concerns, which has driven the development of meat substitutes. Filamentous fungal biomass, commonly known as mycoprotein, is a potential meat substitute since it is nutritious and has filaments to mimic meat fibrils. The current study aimed to investigate the potential use of a cheap substrate derived from the food industry, i.e., residual water in a tempeh factory, for mycoprotein production. The type of residual water, nutrient supplementation, optimum conditions for biomass production, and characteristics of the mycoprotein were determined. The results showed that the residual water from the first boiling with yeast extract addition gave the highest mycoprotein content. The optimum growth condition was a pH of 4.5 and agitation of 125 rpm, and it resulted in 7.76 g/L biomass. The mycoprotein contains 19.44% (w/w) protein with a high crude fiber content of 8.51% (w/w) and a low fat content of 1.56% (w/w). In addition, the amino acid and fatty acid contents are dominated by glutamic acid and polyunsaturated fatty acids, which are associated with an umami taste and are considered healthier foods. The current work reveals that the residual boiling water from the tempeh factory can be used to produce high-quality mycoprotein. [ABSTRACT FROM AUTHOR]