The task of this study was to prepare a complex of tea polysaccharides (TPS) with ferric iron and research its bioavailability in vitro and in vivo. Optimum condition for preparing tea polysaccharides–iron complex (TPIC) was as follows: TPS and FeCl3 with a weight ratio of 1:2.4, reacted in a water bath at 60 °C for 3 h, generating an iron content for TPIC of 14.60 %. The digestion in vitro experiment showed that availability of TPIC was sufficient. Then, iron bioavailability in vivo of TPIC was evaluated by the rat hemoglobin-repletion bioassay with ferrous sulfate (FeSO4) as the positive control. Results indicated that at the end of 21 days’ iron regeneration phase, the values of hemoglobin (Hb) concentration, free erythrocyte protoporphyrin (FEP), serum iron (SI) concentration, and mean cell hemoglobin (MCHC) of rats supplemented with FeSO4 and TPIC increased quickly to those of normal ones. If the bioavailability of FeSO4 was given 100 % with Hb concentration, SI concentration and MCHC as the evaluation index, respectively, bioavailability of TPIC were in the range of 101.85–116 %. These results demonstrate that TPIC is a good iron supplement source for increasing uptake and bioavailability in the body.