Large-scale mariculture of kelp Saccharina japonica will form a large amount of refractory dissolved organic carbon (RDOC) to achieve long-term carbon sequestration. However, the formation process and quantification of RDOC in kelp mariculture are not well understood. The RDOC during the whole kelp mairculture cycle were directly assessed and measured using multidisciplinary analysis technology. The results showed that approximately 50.69–74.66 % of the labile dissolved organic carbon (LDOC) and 14.54–21.51 % of the semi-labile dissolved organic carbon (SLDOC) were gradually degraded over a period of up to 200 days. Actinobacteria, Gammaproteobacteria and Alphaproteobacteria predominantly drove the conversion of LDOC released by kelp to RDOC during the mushroom, adult and mature stages, respectively. In the whole kelp mariculture cycle, the formation amount of RDOC was estimated to be approximately 9.4 g C·ind−1·yr−1, and the formation amount of RDOC accounts for approximately 18.91 % of the carbon storage from kelp biomass. These stable RDOC should be included in the blue carbon budgets of mariculture kelp.