As a promising technology for cancer therapy, cold atmospheric plasma has received extensive attention in recent years. However, previous studies pay more attention on how to achieve and amplify the anticancer range of plasma, while neglecting the clinical needs of precision medicine. Hence there is a lack of small-scale and fixed-point plasma technology for micro-tumor therapy. Here, a 7 μm sized micro-plasma jet device was proposed, and its anticancer effects were evaluated based on three-dimensional (3D) bladder micro-tumor spheroids (T24 cell line) constructed in vitro. The experimental results showed that this micro-plasma jet device can effectively inactivate micro-tumors with diameters ranging from 50 to 300 μm in a short time, and the anticancer effects on 3D T24 cells can still be maintained after irradiation. This work makes the highly precise and efficient inactivation of target micro-tumors possible through plasma technology in clinical therapy.