Fermented soymilk is a traditional oriental food beverage that has various health benefits due to its bioactive compounds. However, the fermentation process and the probiotic strains used can affect the quality and functionality of fermented soymilk. In this study, we designed to develop a novel functional food by fermenting soymilk with Lactobacillus acidophilus (L. acidophilus), a probiotic bacterium that can improve microbiota present in intestine and modulate the immune system. We evaluated the physicochemical, microbiological and sensory attributes of the fermented soymilk, as well as its total phenolic, flavonoids, free amino acids, antioxidant and anticancer activities. The results revealed that L. acidophilus had ability to grow well and produce lactic acid in soymilk, resulting in a pH of 4.4 and a titratable acidity of 0.72 g of lactic acid per 100 mL after 12 h of fermentation. The fermented soymilk had a smooth texture, a pleasant aroma and a sour taste, and was well accepted by the panelists. The fermented soymilk also exhibited a good amount of total phenolic, flavonoids, free amino acids with high antioxidant activity towards DPPH free radicals (IC50 = 55.51 µg/mL) and the inhibition of human colorectal cancer cells (HCT-116) in a dose-dependent manner (IC50 = 67.13 µg/mL). Overall, these findings suggest that fermented soymilk with L. acidophilus is a novel functional food with potential therapeutic values for the prevention and treatment of oxidative stress, cancer and metabolic disorders.