Development of tumor dual mode contrast agents is still a great challenge due to the relative low accumulation at tumor site, which result in the poor imaging efficiency. In this study, we constructed functional technetium-99m (Tc-99m) labeled polyethylenimine (PEI)-entrapped gold nanoparticles (Au PENs) with pH-responsive charge conversion property for enhanced single photon emission computed tomography (SPECT)/computed tomography (CT) dual mode imaging of cancer cells. PEI with amine functional groups (PEI center dot NH2) was successively modified with monomethyl ether and carboxyl functionalized polyethylene glycol (mPEG-COOH), maleimide and succinimidyl valerate functionalized PEG (MAL-PEG-SVA), diethylenetriaminepentaacetic dianhydride (DTPA), and fluorescein isothiocyanate (FI), and used to entrapped gold nanoparticles inside, followed by conjugation with the alkoxyphenyl acylsulfonamide (APAS) through the PEG maleimide, acetylation of the PEI leftover surface amines and Tc-99m labeling. The created nanosystem with the mean Au core diameter of 3.3 nm and with a narrow size distribution displays an excellent colloidal stability and desired cytocompatibility in the investigated Au concentration range. Due to the fact that the attached APAS moieties are responsive to pH, the functionalized Au PENs with a neutral surface charge can switch to be positively charged under slightly acidic pH condition, which could improve the cellular uptake by cancer cells. With these properties, the developed functionalized Au PENs could achieve enhanced dual mode SPECT/CT imaging of cancer cells in vitro. The constructed PEI-based nanodevices may be adopted as an excellent dual mode contrast agent for SPECT/CT imaging of cancer cells of different types.