Sludge has been recognized as a reservoir of antibiotic resistance genes (ARGs) in the wastewater treatment plants. Our previous study has demonstrated that free ammonia (FA, i.e., NH3–N) pretreatment is an effective method for enhancing anaerobic digestion of sludge. However, the effects of FA pretreatment on the removal of ARGs in the anaerobic sludge digestion is still unknown. In this study, several ARGs representing various antibiotic classes and integrase gene (intI1) which is crucial for horizontal transfer of ARGs were chosen. This study demonstrated that combined FA pretreatment (420 mg NH3–N/L for 24 h, under which the highest anaerobic sludge biodegradability was achieved in our previous study) and anaerobic digestion could enhance the removal of aac(6′)-Ib-cr, blaTEM, sul2, tetA, tetB and tetX from sludge by 17–74% compared with anaerobic digestion without FA pretreatment, resulting in a lower ARGs abundance in the anaerobically digested sludge. This is caused by the removal of tested ARGs during FA pretreatment and the reduced abundance of potential microbial hosts of ARGs due to FA pretreatment during anaerobic digestion. The removal of IntI1 was not significantly affected by FA pretreatment and intI1 did not play a large role in the fate of the tested ARGs in this study. This study indicated that FA pretreatment for anaerobic digestion could potentially reduce the spread of ARGs from the sludge to the natural environment during sludge disposal or reuse.