Biofilm formation is one of the defense mechanisms of bacteria against disinfectants and antimicrobials. The aim of this study was to determine biofilm-forming L.monocytogenes from fish processing and salmon surfaces. Biofilm formation at 15, 25, 37, and 40 °C from 1 to 6-days period, adhesion to glass, polypropylene and stainless-steel surfaces, bacterial surface charge and hydrophobicity was determined. Adhesion behavior of the strains was evaluated using Surface Plasmon Resonance (SPR) technique. Totally 32 L.monocytogenes strains belonging to serogroups IIa (n:17), IIc(n:14) and IVb(n:1) were detected from 1320 swabs and 16 smoked salmons. Biofilm formation tests revealed that 21 strains form biofilm on microplate by increasing time and temperature. Although all strains strongly formed biofilm on glass surfaces, two strains slightly adhered polypropylene surfaces. High surface roughness of stainless-steel FeCrNi alloy (Ra = 4.15 nm) and CoCrMo alloy (Ra = 10.75 nm) increased biofilm formation of L.monocytogenes on stainless-steel surfaces. Zeta potential results showed that non-biofilm formers were more negatively charged after 6-days and hydrophobicity couldn't give a distinct distribution among biofilm formers and non-formers. SPR analysis method was evaluated to distinguish biofilm formers to adhere SPR gold chip surfaces. PCR results revealed that all strains were positive for hylA, iap, actA, plcA, plcB, fri, flaA, inlA, inlB, inlC, inlJ, and lmo1386 genes. Additionally, all strains were susceptible to penicillin, ampicillin, meropenem, erythromycin and trimethoprim-sulfamethoxazole. Biofilm-forming, virulence properties of L.monocytogenes strains isolated from fish processing surfaces and smoked salmons were evaluated and SPR was used to differentiate biofilm formers as a sensitive technique for biofilm studies.