Disclosed is a method for detecting a biological signal of a three-dimensional cell culture construct. The method for detecting a biological signal of a three-dimensional cell culture construct includes: providing a three-dimensional cell culture construct that contains at least two cell layers laminated to each other and a sensor particle capable of detecting a biological signal; and observing the sensor particle optically. Preferably, the three-dimensional cell culture construct contains an extracellular matrix including a combination of a protein or polymer having an RGD sequence and a protein or polymer that interacts with the protein or polymer having the RGD sequence, or a combination of a protein or polymer that is positively charged and a protein or polymer that is negatively charged.