The resource recovery of nitrogen and phosphorus in wastewater can be realized based on microalgae biofilm cultivation. Antibiotic from wastewater could potentially transport along the microalgae biofilm and influence microalgae metabolism during the microalgae biofilm-based wastewater treatment technology. Therefore, how one typical antibiotic (sulfamethoxazole, SMX) transport inside algal biofilm was investigated in this study. Furtherly, the effects of SMX on the growth of Chlorella vulgaris and nutrients transfer dynamics along biofilm were studied by microelectrode, Raman spectroscopy and SEM-EDS. The results showed that 5 mg/L SMX could stimulate microalgae photosynthesis and increase the dry weight of microalgae biofilm by 28.56 % on the 30th day. At the same time, the algae density increased by 15.01 %. Sulfur element distribution showed that SMX accumulated 15 % ∼ 25 % more in the middle and bottom layers (40 μm ∼ 140 μm) than in the surface layer of the biofilm. SMX at the deeper layer stimulated the utilization of nitrogen, accelerating the uneven distribution of nitrogen (117 % ∼ 162 % more than the surface layer). 5 mg/L SMX extended the effective photosynthetic region near the surface layer by 40 μm. This change intensified the chemical composition differences between the surface and bottom layers. The correlation analysis showed that nitrogen might be the key factor limiting the growth of microalgae biofilm. This study proved the positive effects of 5 mg/L SMX on microalgae biofilm growth, providing theoretical support for the application of microalgae biofilm technology in antibiotic treatment.