Clostridioides difficile (CD) is a major pathogen responsible of digestive healthcare associated infections. Antibiotic treatments are the main risk factor for CD infections (CDI) because they disrupt the gut microbiota. During dysbiosis, CD spores germinate into vegetative forms which then colonize the gut microbiota, multiply, and produce virulence factors, especially toxin A (TcdA) and toxin B (TcdB). The action of these two toxins will cause an alteration of the cytoskeleton, neutrophils recruitment, thus promoting an inflammatory process, and leading to the clinical manifestations of CDI. The study of toxins is a crucial step in exploring the virulence of this pathogen. Currently, the toxin purification process is either laborious and time-consuming in CD or performed in heterologous hosts. Thus, we propose an easy method to obtain rapidly functional toxins directly using CD as the host. First, we generated two recombinant CD strains with a sequence encoding a His-tag at the 3’- end of the CD 630∆erm tcdA and tcdB genes and then replaced the toxins native promoter with the Ptet promoter inducible by Anhydro-tetracycline (ATc). Then, using these strains, recombinant toxins A (rTcdA) and B (rTcdB) can be produced after induction with ATc and purified using a one-step Ni-affinity chromatography with a simple imidazole elution. Recombinant toxins were compared to native toxins according to cytopathic activity using cell-cytotoxicity-neutralization assay on Vero-cells, and their use as antigens in ELISA assay using Human serums from cured-CDI patients. In conclusion, construction of recombinant strains allowed us to rapidly produce biologically active rTcdA and rTcdB.