•Squid ink Polysaccharide SIP was successfully labelled with fluorescein FTSC.•Squid ink Polysaccharide SIP, as macromolecule, could be transported through epithelial cells.•SIP could be utilized by the host, either by digestive enzymes or gut microbiota, or both. Studies have evidenced that the squid ink polysaccharide (SIP) could enhance intestinal mucosal immunity. To further assess the delivery of SIP through intestinal epithelial cells and determine its bioavailability in the gastrointestinal (GI) tract, the SIP was labelled with fluorescein FTSC. Subsequently, the FTSC-SIP was purified and validated by thin-layer chromatography and liquid chromatography analyses. Based on Caco-2 epithelial monolayer, the transportation of FTSC-SIP was assayed by fluorescence spectrometry. Trace amounts of FTSC-SIP could be transported through the epithelial monolayer. Utilization of FTSC-SIP was evaluated by monitoring the serum FTSC content in mice after FTSC-SIP gavage. FTSC-SIP could be absorbed rapidly, with FTSC showing up in the serum in less than 1 hour after gavage and low molecular weight SIP break-down products in the faeces. Our data imply that SIP can be utilized by the host. This will provide scientific basis for dietary polysaccharide to be used as a functional food ingredient.