Dopamine　participates　in　many　physiological　and　pathological　processes.　Dynamic　monitoring　of　dopamine　levels　in　the　cytoplasm　of　a　single　living　cell　reflects　not　only　the　functional　state　of　dopamine　synthesis　factors　but　also　the　processes　of　related　neurodegenerative　diseases.　Due　to　the　low　content　of　cytoplasmic　dopamine　and　the　difficulty　to　keep　cells　alive　during　the　operating　process,　the　detection　of　cytoplasmic　dopamine　is　still　challenging.　Herein,　a　solid-phase　microextraction　(SPME)　technique　integrated　nanobiosensor　was　employed　to　trace　and　quantify　dopamine　concentration　fluctuations　in　the　cytoplasm　of　a　single　living　cell.　We　designed　a　polypyrrole　modified　carbon　fiber　nanoprobe　as　a　bifunctional　nanoprobe　that　can　extract　cytoplasmic　dopamine　and　then　perform　electrochemical　detection.　This　bifunctional　nanoprobe　can　detect　10 pmol/L　extracted　dopamine　and　detected　a　60%　decrease　of　the　cytoplasmic　dopamine　concentration　in　a　single　living　cell　by　K+　stimulation.　This　study　allowed　for　the　first　time　serially　detecting　cytoplasmic　dopamine　while　keeping　the　target　cell　alive,　which　might　yield　a　new　method　for　research　on　dopamine　neurotoxicity　and　the　related　drug　action　mechanisms　for　neurodegenerative　disease.