DNA barcoding can be utilized as a contemporary tool for species identification and molecular phylogeny. It can decrease many important issues of morphological taxonomy, a small amount of tissue is also required for the identification of species, and the samples can be observed at all stages of growth. DNA barcoding can be utilized in the identification of invasive and endangered species along with conservation biology. In the case of red marine macroalga, DNA barcoding can be helpful for finding high yielding agar as well as for obscure species identification. In the present investigation, the red algae Gracilaria fergusonii J.Ag. was provided from Hare Island in Thoothukudi district, Tamil Nadu, India. DNA was extracted by Cetyl-Trimethyl-Ammonium-Bromide (CTAB) procedure. DNA segments coding for rbcL gene were amplified from total genomic DNA with oligonucleotide primers. The sequencing reaction was carried out in a PCR thermal cycler, utilizing the BigDye Terminator v3.1 cycle sequencing kit. From the present data, the chloroplast marker, rbcL gene is more effective for the DNA barcoding and molecular taxonomy of red marine macroalga.