The development of an effective prophylactic vaccine is still necessary to improve the safety of the conventional although-discontinued smallpox vaccine, and to protect from the threat of deliberate release of variola virus. This need also arises from the number of new cases of animal orthopoxvirus infections each year, and to reduce the risk to animal handlers. Fowlpox (FP) recombinants only replicate in avian species and have been developed against human infectious diseases, as they can elicit an effective immune response, are not cross-reactive immunologically with vaccinia, and represent safer and more promising immunogens for immunocompromised individuals. The aim of this study was the characterisation of two new fowlpox recombinants expressing the A33R vaccinia virus gene either alone (FPA33R) or with the green fluorescent protein (FPA33R-GFP) to verify whether GFP can affect the expression of the transgene. The results show that both FPA33R and FPA33R-GFP can express A33R correctly, but A33R mRNA and protein synthesis are higher by FPA33R than by FPA33R-GFP. Therefore, GFP co-expression does not prevent, but can reduce the level of a vaccine protein, and may affect the protective efficacy of the immune response.