Soil organic matter turnover rates are typically estimated from mass loss of the material over time or from on rates of carbon dioxide production. In the study, we investigated a new way to characterize the concentration-dependent kinetics of amino acids used by measuring microbial uptake and mineralization of 14C-alanine. We measured the depletion from soil solution after additions 14C-alanine. The microbial uptake of 14C-alanine from soil solution was concentration-dependent and kinetic analysis indicated the operation of at least three distinct alanine transport systems of differing affinities. Most of the 14C-alanine depletion from the soil solution occurred rapidly within the first 10–30 min of the incubation after 10 µM to 1 mM substrate additions. At alanine concentrations less than 250 mM, the kinetic parameters for Km and Vmax of the higher-affinity transporter were 60.0 µM and 1.32 µmol g−1 DW soil h−1, respectively. The mineralization of alanine was determined and the half-time values for the rapid mineralization process were 45 min to 1.5 h after the addition at alanine concentrations below 1 mM. The time delay after its uptake into microbial biomass suggested that alanine uptake and subsequent respiration were uncoupled pattern. The microbial N uptake rate was calculated by microbial mineralization, and an estimated Km value of 1731.7±274.6 µM and Vmax value of 486.0±38.5 µmol kg−1 DW soil h−1. This study provides an alternative approach for measuring the rate of turnover of compounds that turnover very rapidly in soil.