[Object] To provide an analysis system, an analysis program, and an analysis method suited for analyzing a 3-dimensional temporal state of an observation object.[Solving Means] An analysis system according to the present technique includes a feature amount calculation section and a reference position calculation section. The feature amount calculation section calculates a feature amount in each of a plurality of moving images of a sample including an observation object, that have been captured at different focal positions, the plurality of moving images having the same phase of movements. The reference position calculation section calculates a reference position of the observation object in a focus direction based on the feature amount.