Background Compared with transistors and small-scale integrated circuits, the total ionizing dose (TID) effect and testing of multifunctional large scale integrated microprocessors are more complex. The difficulty of testing is to analyze the failure mode of microprocessor online from limited information under irradiation. Purpose This study aims to develop an extendable on-line test system for TID effect of microprocessor and carry out preliminary application. Methods The testing system was composed of control circuit, extendable signal acquisition circuit, tested sample interface, upper computer and software. Multiple parametric measurement or functional verification methods such as power consumption current, on-chip memory, communication, clock, analog-to-digital/digital-to-analog converter (ADC/DAC) and direct memory access of microprocessor were provided. Sixteen microprocessors with feature size 40 nm were irradiated with ⁶⁰Co source and tested on-line. Results After the irradiation dose is accumulated to (377.44±20.34) Gy(Si), all samples are malfunctional with digital communication interruption, sudden drop in current consumption, abnormal ADC/DAC output and so on. Conclusion Based on all 12 kinds of parametric measurement or functional verification results, the TID effect of this type of microprocessor is probable to be a functional failure caused by some kernel instructions. The on-line test system of this study can provide more direct data information for the total dose failure mode analysis.