A method of detecting response time-out based on master-slave protocol architecture, which calculates the time required for a slave device to respond to a Modbus request sent by a detection device based on Modbus communication protocol. The method is to send a Modbus request to the slave device through a detection device, and to record the Modbus request sent time, and then to have the slave device provide to the detection device a response. The detection device calculates the response time-out based on the request sent time and the response received time By means of calculating the precise response time-out from the response time-outs which are gotten from the slave device respond to a predetermined number of Modbus requests, the user or manager can determine the response time-out required for the slave device precisely so as to give EXECUTE instruction or command at the accurate time point.