The regulation of cell density is an important segment in microfluidic cell culture, particularly in the repeated assays. Traditionally, the consistent cell density is difficult to achieve owing to the inaccurate regulation of cell density with manual feedback. A novel microfluidic culture method with automatic feedback is proposed for real-time regulation of cell density in this paper. Here, an integrated microfluidic system combining cell culture, density detection and control of proliferation rate was developed. Interdigital electrode structures (IDES) are sputtered on the microchannel for automatically providing the real-time feedback information of impedance. The most sensitive frequency is studied to improve the detection resolution of the sensing chip. Cells were cultured on the chip surface and the cell density was detected by monitoring the alternation of the impedance. The feedback controller is established by least squares support vector machines (LS-SVM). Then, the cell proliferation rate was automatically controlled using the feedback controller to achieve the desired cell density in the repeated assays. The results show that the standard error of this method is 2.8% indicating that the method can keep consistency of cell density in the repeated assays. This study provides a basis for improving the accuracy and repeatability in the further assays of finding the optimal drug concentration.