Oligosaccharides could induce some physiological responses in algae to defend attacks from pathogens. One of the earliest responses induced by oligosaccharides was so-called oxidative burst. This study has surveyed the recognition and oxidative burst response of Porphyra haitanenesis to agaro-oligosaccharide. Oxygen consumption was detected by an oxygen electrode. H2O2 concentrations in the medium were determined by measuring the dimerization of (p-hydroxyphenyl) acetic acid. The elicited oxidative burst was observed by loading the P. haitanenesis with redox-sensitive fluorescent probe dichlorohydrofluorescin (DCFH-DA). Gene expression level of NADPH oxidase (named Phrboh) was detected by real-time PCR. 2-AMAC labeled agaro-oligosaccharides were used to research the binding of agaro-oligosaccharide on algae. Results showed that two respiration peaks were observed after addition of 100 μg/mL agaro-oligosaccharide in P. haitanensis. Fourfold increase occurred in the first respiration peak 4 minutes after the addition, and fourteen times higher in the second response 10 minutes after the exposure to agaro-oligosaccharide. The accumulation of H2O2 in the culture medium was detected 5 minutes after challenged with agaro-oligosaccharide. The concentration of H2O2 reached its peak after 15 minutes, which was nearly ten times higher than control. The gene expression level of NADPH oxidase in P. haitanensis was up-regulated 3 minutes after the addition of agaro-oligosaccharide, suggesting that the synthesis of H2O2 was closely related to the activation of Phrboh. Based on the analyses of the fluorescence of redox-sensitive dye, activated oxygen species mainly accumulated around the plasma membrane. Under the fluorescent microscope, specific binding of 2-AMAC labeled agaro-oligosaccharide on cell plasma membrane was also observed, which indicated that there was recognition site of agaro-oligosaccharide on P. haitanensis plasma membrane. In summary, P. haitanensis could recognize agaro-oligosaccharide and respond with oxidative burst which was associated with the activation of NADPH oxidase.