Bioluminescence is interesting, among other reasons, for the various technological applications that have been derived from it. Among these applications, developing visualization techniques to record the expression of one or more genes simultaneously in real-time are particularly useful. With this in mind, this study aimed to generate a recombinant Pyrocystis lunula luciferase protein (Luci D2-3 partial CDS). As the main results, i) a fragment of 1467 bp of the luciferase (LCFb) mRNA of the dinoflagellate P. lunula, containing part of domain 2 and all of the domain 3, was cloned in the pET28a vector; ii) the constructed vector was used to transform Escherichia coli to express the recombinant protein and subsequently purify it through an affinity chromatography procedure using a His-Tag; and iii) the purified protein (~50 kDa) was further analyzed by mass spectrometry to confirm its identity. Despite being unable to perform activity tests with the luciferin substrate, the evidence from previous studies indicates that the recombinant protein obtained in this case is enzymatically active. Due to the limited number of currently available luciferases, synthesizing this recombinant protein represents a useful tool, especially in designing expression assays coupled to multiple reporter genes, thus expanding the palette of proteins available for developing this type of biotechnological advances.