Microorganisms are present as either biofilm or planktonic species in natural and engineered environments. Little is known about the selection pressure emanating from exposure to sub-minimal inhibitory concentration of antibiotics on planktonic vs. biofilm bacteria. In this study, an E. coli bioreporter was used to develop biofilms on glass and high-density polyethylene (HDPE) surfaces, and compared with the corresponding planktonic bacteria in antibiotic resistance expression when exposed to a range of μg/L levels of tetracycline. The antibiotic resistance-associated fluorescence emissions from biofilm E. coli reached up to 1.6 times more than those from planktonic bacteria. The intensively developed biofilms on glass surfaces caused the embedded bacteria to experience higher selection pressure and express more antibiotic resistance than those on HDPE surfaces. The temporal pattern of fluorescence emissions from biofilm E. coli was consistent with the biofilm-developing processes during the experimental period. The increased expression of antibiotic resistance from biofilm bacteria could be attributed to the high affinity of tetracycline with extracellular polymeric substances (EPS). The enhanced accumulation of tetracycline in biofilms could exert higher selection pressure on the embedded bacteria. These results suggest that in many natural and engineered systems the higher antibiotic resistance in biofilm bacteria could be attributed partially to the retention antibiotics by the EPS in biofilms.