During tempering of martensite a complex carbide precipitation sequence appeared in the steel particularly containing microalloyed elements such as V, Nb and Ti. The alloy carbide, which usually precipitates following cementite precipitation in certain temperature range, has been designed to maximize the number density and to retard the coarsening for increasing soften resistance. During the nucleation stage of the alloy carbide, the dislocations and interfaces of distinct phases are the actively precipitated position. However, because of extremely small sizes, their characterization is restricted by the analytic resolution of conventional methods. The 3D atom probe (3DAP) is a particularly helpful instrument with atomic spatial resolution and high componential sensitivity in the characterization of the early stages of precipitation reactions. In this paper, the 3DAP companied with TEM and micro-hardness test was applied to characterize the early nucleation stage of the alloy carbides precipitated during tempering of Nb-V microalloyed steel after quenched from solution treatment at 1200 ℃ for 0.5 h. With the tempering time prolonged from 0.5 to 100 h at 450 ℃, the micro-hardness of the experimental steel changes with the microstructure recovery and carbide evolution (from cementite to alloy carbide). The two peak hardness values appeared at 4 and 100 h tempering are related to precipitate cementite and alloy carbide, respectively. The nucleation of the alloy carbides happens during 30 h tempering at 450 ℃. The alloyed elements dynamically redistributed in the existed remnant austenite, that is, non-carbide-forming elements such as Si and Al diffuse to matrix from the cementite, whereas the carbide-forming elements such as Mo, Nb and V enriched in the cementite, resulting in in situ transformation of alloy carbides. The intragranular defects such as high density dislocation in martensite also act as nucleation sites of alloy carbide, at which V and Nb directly combine with C and lead to the formation of G.P. zone before formation of alloy carbides. Besides, the interfaces of the remnant austenite/matrix and the undissolved AlN/matrix are also energetically favorable nucleation sites, resulting in heterogeneous nucleation of alloy carbides. With the decrease of dislocation density and the dissolutions of cementite and remnant austenite, the consumption of the potential nucleation sites ends the nucleation stage of alloy carbide when tempering for 100 h.