Highly fluorescent surface modified polyacrylonitrile nanoparticles (PAN NPs) of 50 nm diameter were fabricated for selective Cu(2+) sensing. After surface modification, the PAN NPs were converted to amidine/Schiff base dual-modified PAN nanoparticles (tPAN NPs) with a Cu(2+) sensing property and high QY (0.19). The selectivity of tPAN NPs for Cu(2+) is much higher than that of other metal ions due to the fact that amidine group on the surface of tPAN NPs has a higher binding affinity with Cu(2+). The effect of other metal ions on the fluorescence intensity of the tPAN NPs was also studied, and other metal ions showed a low interference response in the detection of Cu(2+). Furthermore, as a metal ion chelator, ethylenediaminetetraacetate can competitively interact with Cu(2+) to recover the quenched fluorescence of tPAN NPs. The tPAN NPs are easily introduced into cells and exhibit low toxicity, enabling their use as a fluorescence sensor for Cu(2+) in living cells. The tPAN NPs provide a new direction for the development of copper ion sensors in living cells.