Abstract: This study was designed to research the response of free-living conchocelis growth and sporangium branches formation stage of free-living conchocelis of Pyropia haitanensis to agaro-oligosaccharide. After being treated with agaro-oligosaccharide, the net photosynthetic rate of free-living conchocelis of P. haitanesis was measured by liquid phase electrode; the concentration of H₂O₂ in the medium was determined by the dimerization of (p-hydroxyphenylethyl) acetic acid; the floridoside content was detected by LC-MS; the H₂O₂ related genes (Phrboh, PhSOD) and the floridoside synthesis related genes (Phnho1, Phgpdh, Phtps) expression was detected by real-time PCR; the number of sporangium branches was examined by microscopy. The results showed that the agaro-oligosaccharides can stimulate a series of responses of free-living conchocelis of P. haitanesis. These responses include increased photosynthetic rate; increase the content and biosynthesis of photosynthetic products of floridoside; the release of H₂O₂ continues to increase which was associated with the activation of two enzymes associated with H₂O₂ gene Phrboh and PhSOD. In addition, agaro-oligosaccharides can also promote the development of free-living conchocelis of P. haitanesis. On thirtieth day of culture, the sporangium branches formation rate of agaro-oligosaccharides treatment group reached 59%, significantly higher than the control group 46%. In conclusion, agaro-oligosaccharide can increase the free-living conchocelis of P. haitanesisphotosynthetic rate and photosynthetic products, and can increase the expression of H₂O₂ synthesis enzyme to induce the release of ROS. Meanwhile, agaro-oligosaccharides can promote the reproduction of free-living conchocelis of P. haitanesis.