In this report, we describe a cloning procedure for gene replacement by double homologous recombination in Plasmodium yoelii, which requires only one digestion and ligation step. This significantly shortens the time required to complete the production of the targeting vector. Furthermore, for more efficient phenotypic evaluation of the gene knockout parasites, we have also introduced a fluorescent protein cassette into the targeting vector. This allows for a more rapid assessment of parasite growth in all of its developmental stages. In addition, the introduction of the fluorescent marker via the replacement strategy confers the stable integration of the marker.