Abstract: According to principle that writing a Boot Loader program in single-chip microcomputer can realize on-line self-programming updating of ladder diagram of PLC by user, the paper introduced a design of Boot Loader program of ATmega128 based on PLC application. The program writes object code of ladder diagram of PLC into FLASH of single-chip microcomputer through RS232 interface with imitating mitsubishi communication protocol. The experiment showed that it makes design of PLC application software be more flexible and improves development efficiency that applying Boot Loader in PLC to realize downloading and updating of ladder diagram.