A dynamic flow-through microcolumn extraction system based on extractant recirculation is herein proposed as a novel analytical approach for simplification of bioaccessibility tests of trace elements in sediments. On-line metal leaching is undertaken in the format of all-injection (AI) analysis, which is a sequel of flow injection analysis, but involving extraction under steady-state conditions. The minimum circulation times and flow rates required to determine the maximum bioaccessible pools of target metals (viz., Cu, Zn, Cd, and Pb) from lake and river sediment samples were estimated using the Tessier's sequential extraction scheme and an acid single extraction test. The on-line AIA method was successfully validated by mass balance studies of CRM and real sediment samples. Tessier's test in online AI format demonstrated to be carried out by one third of extraction time (6 h against more than 17 h by the conventional method), with better analytical precision (< 9.2% against > 15% by the conventional method) and significant decrease in blank readouts as compared with the manual batch counterpart.