将主窗口代码替换class MyWindow(QMainWindow):

from PyQt5.QtWidgets import QApplication, QMessageBox, QMainWindow
from PyQt5.QtCore import QSharedMemory, QProcess
import sys
import os
import pygetwindow as gw


class SingleApplication(QApplication):
    def __init__(self, argv=[]):
        super(SingleApplication, self).__init__(argv)
        self.window = None
        self.mem = QSharedMemory("YourAppUniqueString")  # 应用程序唯一字符串
        self.is_running = self.mem.attach()
        if self.is_running:
            self.mem.detach()

    def check_is_running(self):
        if self.is_running:
            # 获取已经运行的窗口句柄
            hwnd = gw.getWindowsWithTitle(app_title)[0]._hWnd
            # 恢复窗口
            gw.Window(hwnd).restore()
            # 激活窗口
            gw.Window(hwnd).activate()
            # 关闭此次程序
            os.system("taskkill /F /PID %s" % os.getpid())
            return True
        return False

    def run(self, window):
        if not self.is_running:
            self.mem.create(1)
            self.window = window
            self.window.show()


class MyWindow(QMainWindow):
    def __init__(self):
        super().__init__()
        self.setWindowTitle(app_title)


if __name__ == "__main__":
    app_title = 'My Window'
    app = SingleApplication(sys.argv)
    if not app.check_is_running():
        window = MyWindow()  # 你的主窗口类
        app.run(window)
    sys.exit(app.exec_())