Sulfated polysaccharides (SP) are widely used as industrial additives and pharmaceutical intermediates. As SP can only be extracted from sea algae, making them scarce raw materials. Recently, SP have been detected and extracted from the waste activated sludge of a saline secondary wastewater treatment plant, suggesting that there are alternative primary producers and synthesis pathways of the SP within the biological activated sludge. This study aimed to identify the primary SP producers, the SP biosynthesis pathways as well as the SP production rates in different types of activated sludges cultivated anoxically and/or anaerobically, with and without the presence of sufficient sulfate. The results showed that alternating anaerobic/anoxic conditions in sludge effectively produced the SP by the ordinary heterotrophic organisms (OHOs). The synthesis pathways for the three most common bioactive SP viz. fucoidan, carrageen, and heparin, were identified and elucidated at both the substrate and enzymatic levels. The Western Blot analyses revealed key enzymes for the SP synthesis (e.g., GDP-L-fucose-synthetase, GDP-fucose-pyrophosphorylase, β-1,4-galactosyltransferase), when sulfate was sufficient (>170 mg S/L) under an alternating anaerobic/anoxic conditions. In contrast, the absence of sulfate suppressed the SP production during the initial step of the SP generation. The synthesis of the SP in the sulfate-reducing (anaerobic) sludge was suppressed by the enzymatic inhibition, when sulfide exceeded 160 mg S/L, due to the competition for energy between the SP synthesis and sulfide detoxification. However, in the case of the sulfide-oxidizing sludge both the organic carbon and metabolism energy deficiencies inhibited the SP production. The findings of this study expand the understandings of the SP synthesis in the activated sludge under different operating conditions, including different sulfate levels.