Florfenicol (FFC) as a chloramphenicol's derivative is a special broad-spectrum antibiotic that was used in veterinary clinics. In the present study, we investigated the effect of different doses of FFC on the humoral immune response of broiler chickens to Newcastle disease virus (NDV) vaccine under the impact of E. coli infection. In addition, the expression of the interferon-inducible genes (IRF7, 2'-5'OAS and Mx1) were analyzed in the spleen tissue of these chickens using quantitative real-time PCR (qRT-PCR). The non-treated group with FFC and non-infected with E. coli had the highest immune responses against NDV compared with the FFC treated groups. In the case of E. coli infection, the group treated with FFC (30 mg/Kg BWt) showed lower NDV HI and IgG ELISA Ab levels compared to the group treated with FFC (60 mg/Kg BWt). A dose dependent up-regulation was observed in the level of the interferon-alpha pathway related genes (IRF7 and 2'-5'OAS) in the FFC treated groups compared to the non-treated group. At the slaughter time, the numbers of adipocyte in the bone marrow were significantly higher with moderate atrophy of the hematopoietic lineages in the FFC treated birds compared to the non-treated birds. These results indicated that this FFC dosage dependent increase in the humoral immune responses against NDV vaccine could be attributed to its efficient therapeutic effect on the E. coli infection. However, the increase in the FFC dosage can negatively but temporarily affect the chicken body weights. Additionally, it can exert up regulation effect on the chicken innate immune response with moderate hypoplasia of the bone marrow cells.