Abstract: In order to establish the standard preparation methodology for bioactive polysaccharide of fucoidan, this study carried out the research of preparation method of fucoidan from Saccharina japonica. Three extraction methods, i.e. high temperature pressure extraction (HPE), acid extraction (AE), and microwave extraction at 400 W (M400W), 500 W (M500W), and 600 W (M600W), were selected. Subsequently, two precipitation methods, i.e. calcium chloride precipitation (CP) and ethanol precipitation (GAP) were used to remove alginate followed by finally precipitation of fucoidan with high concentration ethanol. Fucoidan extracts prepared in totally ten different conditions were collected, of which the contents of total sugar, total phenols, protein, uronic acid, and sulfate groups were analyzed. Meanwhile their antioxidant activities including Fe³⁺ reduction ability, ABTS⁺ and DPPH free radical scavenging ability were determined. The correlation analysis was conducted based on the above determined results to discover the relationship between preparation methods and fucoidan qualities. Accordingly, the preparation methods for fucoidan of high yield, high purity, and excellent antioxidant activities were selected. Results showed that the yields of fucoidan by acid extraction with calcium precipitation (AE-CP) and microwave extraction with calcium precipitation (M400W-CP, M500W-CP, M600W-CP) were 3.821%, 3.516%, 3.376%, and 2.998%, respectively. Meanwhile, the sulfate content was 17.734%, 23.872%, 23.376%, and 22.149%, respectively. Their protein and total phenol content were below 1%, the uronic acid content was below 10%, and the Trolox equivalent (ABTS⁺ radical scavenging ability) was 5.637, 5.336, 6.567, and 5.616 μg/mg, respectively. Thus, the above four preparation conditions were ideal for processing of fucoidan, among which the method of microwave extraction followed by calcium precipitation showed advantages of short processing time and low solvent consumption that met the requirement of the environmental-friendly processing. This study systematically investigated the effects of preparation methods on the yield and quality of fucoidan, which provided a practical preparation methodology for fucoidan from Saccharina japonica with high yield, high purity, and high antioxidant activity.