OBJECTIVE Surface electromyography (sEMG) signal decomposition is of great importance in examining neuromuscular diseases and neuromuscular research, especially dynamic sEMG decomposition is even more technically challenging. METHODS A novel two-step sEMG decomposition approach was developed. The linear minimum mean square error estimation was first employed to extract estimated firing trains (EFTs) from the eigenvector matrices constructed using the non-negative matrix factorization (NMF). The firing instants of each EFT were then classified into motor units (MUs) according to their specific three-dimensional (3D) space position. The performance of the proposed approach was evaluated using simulated and experimentally recorded sEMG. RESULTS The simulation results demonstrated that the proposed approach can reconstruct MUAPTs with true positive rates of 89.12 ± 2.71%, 94.34 ± 1.85% and 95.45 ± 2.11% at signal-to-noise ratios of 10, 20, and 30 dB, respectively. The experimental results also demonstrated a high decomposition accuracy of 90.13 ± 1.31% in the two-source evaluation, and a high accuracy of 91.86 ± 1.14% in decompose-synthesize-decompose- compare evaluation. CONCLUSIONS The adoption of NMF reduces the dimension of random pattern under the restriction of non-negativity, as well as keeps the information unchanged as much as possible. The 3D space information of MUs enhances the classification accuracy by tackling the issue of relative movements between MUs and electrodes during dynamic contractions. The accuracy achieved in this study demonstrates the good performance and reliability of the proposed decomposition algorithm in dynamic surface EMG decomposition. CONCLUSIONS The spatiotemporal information is applied to the dynamic surface EMG decomposition.