The aims of the present study are to isolate and characterise structure TLR3 and IL1β gene and evaluate the potential and signaling mechanism following exposure of Polyinosinic: polycytidylic acid (poly (I:C)), Lipopolysaccharide (LPS) and Zymosan as antigens in gouramy (Osphronemus gouramy). Gouramy kidney cells were stimulated with LPS, Poly I:C and Zymosan. Following incubation at 28°C, relative expression levels of Toll Like Receptor 3 (TLR3) and interleukin-1β (IL-1β) were examined at one hour and six hour after treatment. A Real Time Polimerase Chain Reaction approach was utilised to search for the effects of Poly I:C, LPS, and Zymosan exposure to gouramy kidney cells between one hour to six hours after treatment, LPS in kidney cell increased expression of interleukin-1β and downregulated expression of TLR3. Poly (I:C) which is an antigen that responds to antiviral, induces an increase in the transcription both of Toll like receptor 3 and IL-1β. Zymosan in kidney cells increased expression of TLR3 but downregulated expression of IL1β. This study shows that TLR3 was activated not only by Poly I: C but also LPS and Zymosan. However, antigen Poly IC-induced labor IL-1β and TLR3 expressed higher than the antigen LPS and Zymosan, so, this research showed a TLR3 response to Poly I:C is more dominant than Zymosan and LPS. TLR3 in gouramy kidney cell transcript was identified in response to poly I:C, Zymosan, and LPS. These results indicate that TLR3 in gouramy also plays a role for defense against bacterial infection and virus.