Nuclear proteins are crucial in cells and are greatly linked to various biological functions. Abnormal expression of nuclear proteins is associated with many diseases ranging from inflammation to cancer. However, it remains challenging to detect nuclear proteins in single cells because of their low abundance and complex subcellular environment. Herein, we report a subcellular-resolved plasmonic immunosandwich assay (srPISA), for probing nucleus-enriched proteins in single living cells with minimal disruption. We demonstrated the specific extraction and ultrasensitive detection capabilities of the srPISA by probing low-copy-number nuclear telomerase in single living cells and further compared the telomerase expression levels in these single cells. Additionally, we showed the subcellular resolving capability of the srPISA by probing the spatial distribution of smad2 in the nucleus and cytoplasm of single living cells. We found that smad2 was expressed both in the nucleus and the cytoplasm, but showed different expression levels. Moreover, smad2 distributed more homogeneously in the nucleus than in the cytoplasm. Finally, the srPISA of nuclear telomerase in cell division strongly verified that the subcellular analytical results obtained by the srPISA are reliable. Overall, the srPISA approach allowed specific extraction and ultrasensitive detection of target low-copy-number proteins at the subcellular level, providing a unique and powerful single cell analysis tool for cell biology studies.