In this work, we report the coupling of solid-phase microextraction (SPME) enabled by surface-coated acupuncture needles with nano-electrospray mass spectrometry (nanoESI-MS) for the analysis of bioactive molecules in living plants. The needle tip was oxidized by a mixture of nitric acid and hydrogen peroxide solution and then subject to surface coating via carbonization of paraffin. A combination of oxidation and surface coating resulted in a thin coating of carbon film, whereby the significantly increased surface area promoted both analyte enrichment and ionization for MS analysis. The analytical performances were evaluated through the characterization of small molecules, peptides and proteins. Compared with conventional nanoESI, our new strategy of employing surface-coated needles had a high salt tolerance. The streamlined experimental workflow could be completed within one minute. The linear dynamic ranges for L-histidine and L-lysine, as two representatives, were over two orders of magnitude with a limit of detection (LOD) of 3.0~5.0 ng/mL. A mark is made on the needle at 2 mm from the tip, the needle is then kept in the sample for 30 s. In vivo sampling and identification of -tomatine and organic acids from the stem of a living tomato plant were demonstrated as a practical application, while the physiological activities of the plant were not disrupted due to the minimally invasive sampling. We anticipate that the developed strategy may be of potential use for real-time clinical and other on-site analyses.