Antibiotic resistance genes (ARGs) have been identified as serious threats to public health. Despite the widespread in various systems, dynamics of ARGs in three-dimensional multifunctional biofilm (3D-MFB) treating greywater are largely undefined. This work tracked the distributions and dynamics of eight target genes (intI1, korB, sul1, sul2, tetM, ermB, blaCTX-M and qnrS) in a 3D-MFB during greywater treatment. Results showed that hydraulic retention times at 9.0 h achieved the highest linear alkylbenzene sulfonate (LAS) and total nitrogen removal rates at 99.4% and 79.6%, respectively. ARGs presented significant liquid-solid distribution feature, but non-significant with biofilm position. Intracellular ARGs (predominant by intI1, korB, sul1 and sul2) at bottom biofilm were 210- to 4.2 × 104- fold higher than that in cell-free liquid. Extracellular polymeric substances (EPS)-attached LAS showed linear relationship with most of ARGs (R2 > 0.90, P < 0.05). Sphingobacteriales, Chlamydiales, Microthrixaceae, SB-1, Cryomorphaceae, Chitinophagaceae, Leadbetterella and Niabella were tightly bound up with target ARGs. Key is that EPS-attached LAS considerably determines the occurrence of ARGs, and microbial taxa play an important role in the dissemination of ARGs in the 3D-MFB.