Large-scale vortical structure of a turbulent separation bubble affected by unsteady wake is essential to understand flow mechanisms in various fluid devices. A spoked-wheel type of wake generator provides unsteady wake, which modifies the turbulent separation bubble significantly by changing rotation directions and passing frequencies. A detailed mechanism of vortex shedding from the separation bubble with unsteady wake is analyzed by taking a conditional average with spatial box filtering, which spatially integrates measured signals at pre-determined wavelength. A convecting nature of the large-scale vortical structure is analyzed carefully. Spatial evolution of the large-scale vortical structure with frequency variance is also exemplified.