One of the cost-effective approaches to reduce the prevalence of iron deficiency anaemia is by developing iron fortified food. However, adding iron directly into food will not only affect the organoleptic quality of the food, but will also reduce the effectiveness, stability and bioavailability of the iron added. Chitosan as linear polysaccharide, that is biodegradable and biocompatible, can be used as a protective wall material to encapsulate iron by adding tripolyphosphate (TPP) as the crosslinking agent. The aims of this study were to formulate an extended release system of chitosan microspheres loaded with iron gluconate using spray drying method. The physicochemical characteristics and the stability of iron microspheres in simulated gastrointestinal fluids were also evaluated. Various concentrations of TPP (0%, 1%, 2%, 3% w/v) were used for this investigation. The results indicated that the yield, loading capacity, encapsulation efficiency, particle size, particle morphology, and release profile of iron microparticles were influenced by the interaction of chitosan and TPP as cross linking agent. The mean iron loading, maximum yield, and encapsulation efficiency were 2.51%, 43.47%, and 38.19%, respectively. Preparation of iron microparticles using spray drying method resulted in smooth spherical particles with the average diameter ranged from 1.000-12.554μm. The increase concentration of TPP formed larger size of particles. Furthermore, the release analysis of microparticles loaded iron was conducted using synthetic gastric fluids (SGF) and synthetic intestine fluids (SIF) for 2 and 4 hours, simultaneously. The burst release profile was obtained in the absence of TPP, while the controlled profile was shown in the presence of TPP. Increasing amount of TPP in the solution reduces the release in the first 1-2 hours, as well as at the end of the 7 hour period of release. Using TPP 3% the release in one hour reached 50% and about 60% in 7 hours. Compared to chitosan solution without TPP, the release in one hour reached 80% and in 7 hours it was 100% release. The presence of TPP is thus an important factor to be addressed when preparing iron-loaded chitosan microspheres with extended release characteristics.