We studied the effects of initial pH, different nitrogen sources, and cultivation methods (shake flask and static culture) on biomass production, exopolysaccharides (EPS), and adenosine by Paecilomyces tenuipes. Relatively low pH levels were optimal for mycelial growth and EPS production. Yeast extract was the most effective organic nitrogen source for EPS production, whereas soybean extract was the best for adenosine production. A high C/N ratio was beneficial for adenosine production; however, excessively high C/N ratios reduced adenosine production. Static fermentation significantly increased adenosine production. A Box-Behnken design was used to optimize adenosine production; the optimal conditions for adenosine production by P. tenuipes were pH 7.0, soybean concentration of 3%, and a static culture period of 20 days, with the maximum adenosine production of 141.10 mg/L (predicted value: 128.05 mg/L).