Single cell protein (SCP) provides an alternative protein source to partially replace the conventional agricultural resources and support the increased nutritional needs. Inexpensive feeding source is one of the key limiting factors for the expansion of SCP production. The present study examined the valorization of biogas derived from the anaerobic digestion (AD) of sewage sludge and the discarded effluent as nutrients source to produce SCP using methanotrophic bacteria. Results indicated that the mixed methanotrophic culture can grow well on the pasteurized AD supernatant and biogas, succeeding in promising dry weight (DW) yield (0.66 ± 0.01 g-DW/g-CH4 and 11.54 ± 0.12 g-DW/g-NH4+). Methylomonas (56.26%) and Methylophilus (24.60%) spp. were the two main representatives of the mixed culture. The produced dried biomass had a protein content higher than 41% w/w, including essential amino acids like histidine, valine, phenylalanine, isoleucine, leucine, threonine and lysine. The cultivated SCP shows potential utilization as protein source for animal diets.