The study of fluorescent polydopamine nanoparticles (F-pDA NPs) for the purpose of sensing and cell-staining has been investigated over the past decade. However, most of general synthesis strategies include potentially dangerous process or need to use cytotoxic chemicals that restrict the application range. Inspired by the adhesive structures in mussel chemistry, we employ glutathione (GSH) as a stabilizing agent to assemble the fluorescent GSH-integrated polydopamine nanoparticles (F-GSH-pDA NPs) via a straightforward one-pot oxidation method. In contrast to common methods that rely on H2O2 or specific catalyst, this novel way has been carried out through a single step based on only adjusting the pH value of GSH/Dopamine solution. The photoluminescence results demonstrated the possibility of controlling the emission wavelength from 480 to 540 nm by simply changing the excitation wavelength (from 320 to 450 nm). In particular, the emission profile shifted towards the longer wavelength region (from 493 to 525 nm) in the presence of acidic environment (pH 6 to 1). This blue-green luminescence could be switched off by the addition of Cu2+ or Fe3+ and the detection limits were determined to be 0.73 mu M and 0.66 mu M respectively. Further discrimination between Cu2+ and Fe3+ could be realized by absorption spectra changes. For the first time, the intracellular delivery of the functional nanoplatform has been reported and Cu2+ or Fe3+ recognition could be performed in two adherent cell lines (U937 and HeLa cells).