Bacillus natto is commonly used in industrial production of menaquinone-7, an important vitamin which plays a crucial role for blood clotting and may contribute to prevention of cardiovascular disease and osteoporosis. This study determined the optimal combination of key nutrients and established an effective use of surfactant in a coupling medium to enhance the yield of extracellular MK-7. MK-7 yield of 31.18 mg/L was achieved under optimal conditions containing 53.6 g/L glycerol, 100 g/L soy peptone, and 10 g/L K2HPO4. A maximal yield of 40.96 mg/L MK-7 and a secretion ratio of 61.1% were obtained when 20 g/L soybean oil was supplemented at the logarithmic phase. The non-ionic surfactant span 20 was the second most promising surfactant in improving product yield, whereas addition of 2 g/L betaine exerted a minimal effect on secretion ratio of MK-7 at 19.1%. The results collectively showed that the supplementation of surfactants was an effective strategy to regulate cytomembrane permeability.