This study reported a new, renewable and high-efficient strategy for anaerobic fermentation, i.e., using free ammonia (FA) to pretreat waste activated sludge (WAS) for 1 d and then combining with rhamnolipid (RL), by which the short-chain fatty acids (SCFA) production was remarkably improved. Experimental results showed the maximal SCFA production of 324.7 ± 13.9 mg COD/g VSS was achieved at 62.6 mg FA/L pretreatment combined with 0.04 g RL/g TSS, which was respectively 5.95-fold, 1.63-fold and 1.41-fold of that from control, FA pretreatment and RL pretreatment. Mechanism investigations revealed that FA + RL enhanced sludge solubilization and hydrolysis, providing more organics for subsequent SCFA production. It was also found that the combined method inhibited acidogenesis and methanogenesis, but the inhibition to methanogenesis was much severer than that to acidogenesis. Finally, the feasibility of NH4+-N and PO3−4-P, released in fermentation liquor, being recovered as magnesium ammonium phosphate (MAP) was confirmed.