A novel combined process, consisting of thermal-alkali pretreatment, enzymatic hydrolysis, and anaerobic digestion (AD) was utilized to methanize corn straw (CS) and cattle manure (CM) efficiently. This study aimed to identify the carbon/nitrogen (C/N) ratio of the mixed hydrolysates of CS and CM that maximized methane production in an AD reactor. Additionally, pretreatment conditions for CS and CM were evaluated. The optimum condition of pH 10 was 80 °C and 3 h of thermal-alkali pretreatment to produce 42% of hydrolysis efficiency, while a further enzymatic process increased the efficiency to 72%. The C/N ratio was optimized during the co-digestion of the mixed hydrolysates, and better performances were obtained with a C/N ratio of 11 to 30 having specific methane production from 180 to 280 mL/g CODadded. The maximum methane production reached to 280 mL/g CODadded at the C/N ratio of 20. Approximately 75% of the total organic matter from the liquid fractions of mixed hydrolysates was converted to methane. Trace elements in CM hydrolysates may also promote the methane yield. This community structure change was proposed to be an internal response for different C/N ratio adaptation. An inappropriate C/N ratio may cause accumulation of free ammonia or volatile fatty acids, which would inhibit methanogens, but not affect the acidogens.