Micronutrient deficiency is commonly demonstrated in farmlands of fluvo-aquic soil, a typical soil type in the Huang-Huai-Hai Plain of China. The objectives of this study were to explore effects of different long-term fertilization practices on balance of micronutrients in the soil, and changes of soil micronutrients in fraction using the BCR sequential extraction method. A field experiment, designed to have 7 treatments, i.e. OM (Organic Manure), 1/2OM+1/2 ( Fertilizer)NPK, NPK, NP, PK, NK, and CK (No Fertilizer), was laid out and started in 1989. After more than 20 years of fertilization, various extents of accumulation of micronutrients were observed in the surface layer soil (0～20cm), and found to be greatly affected by their transfer between different soil layers. Results of sequential extraction show that the contents of soil available (DTPA-micronutrient) Fe, Cu and Zn were higher, while the content of soil available Mn was relatively lower than the critical levels in the other alkalic soils. The Residue-Fractions of soil Fe (>90%), Cu (>65%) and Zn (>70%) accounted for a high proportion of their respective total. Interestingly, DTPA, acid soluble and oxidizable fractions of soil micronutrients accumulated more with the increasing soil organic matter level, which was probably the driving factor for the changes of soil micronutrients in fraction. Long term application of K fertilizers also increased DTPA and acid soluble fractions of soil micronutrients, but decreased the total of Fe and Mn in the surface soil layer, while long-term application of P fertilizers decreased the contents of DTPA and acid soluble fractions of soil micronutrients through precipitation, but increased the totals of Cu and Zn.