Abstract: A manufacturing method of separating and purifying neoagarooligosaccharides having degrees of polymerization of 2-22 includes the steps of adding crude enzyme solutions with agarases produced by Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108, respectively, into an agar polysaccharide extract solution, and obtaining a neoagarooligosaccharides solution after a hydrolysis on the algal polysaccharide (such as agar) is performed; performing a separation to the neoagarooligosaccharides solution by a ultrafiltration (UF) system to obtain a neoagarooligosaccharide eluent; performing a separation to the neoagarooligosaccharide eluent by a semi-preparative high-performance liquid chromatography system equipped with a molecule size exclusion chromatography function to obtain the neoagarooligosaccharides having the degrees of polymerization from 2 to 22; and using a fraction collector to collect the neoagarooligosaccharides, and obtaining purified single neoagarooligosaccharide products with different mo