The nucleus is delineated by the nuclear envelope (NE), which is a double membrane barrier composed of the inner and outer nuclear membranes as well as a ~40 nm wide lumen. In addition to its barrier function, the NE acts as a critical signaling node for a variety of cellular processes which are mediated by protein complexes within this subcellular compartment. While fluorescence fluctuation spectroscopy (FFS) is a powerful tool for characterizing protein complexes in living cells, it was recently demonstrated that conventional FFS methods are not suitable for applications in the NE because of the presence of slow nuclear membrane undulations. We previously addressed this challenge by developing time-shifted mean-segmented Q (tsMSQ) analysis and applied it to successfully characterize protein homo-oligomerization in the NE. However, many NE complexes, such as the linker of the nucleoskeleton and cytoskeleton (LINC) complex, are formed by heterotypic interactions, which single-color tsMSQ is unable to characterize. Here, we describe the development of dual-color (DC) tsMSQ to analyze NE hetero-protein complexes built from proteins that carry two spectrally distinct fluorescent labels. Experiments performed on model systems demonstrate that DC tsMSQ properly identifies hetero-protein complexes and their stoichiometry in the NE by accounting for spectral crosstalk and local volume fluctuations. Finally, we applied DC tsMSQ to study the assembly of the LINC complex, a hetero-protein complex composed of Klarsicht/ANC-1/SYNE homology (KASH) and Sad1/UNC-84 (SUN) proteins, in the NE of living cells. Using DC tsMSQ, we demonstrate the ability of the SUN protein SUN2 and the KASH protein nesprin-2 to form a hetero-complex in vivo. Our results are consistent with previously published in vitro studies and demonstrate the utility of the DC tsMSQ technique for characterizing NE hetero-protein complexes.