As an effective nuclear energy resource, uranium plays an important role in industry and energy but the wastes of uranium also cause radioactive contamination, which is harmful to the environment and the human body. Herein, a responsive photonic crystal (PC) film sensor for the ultrasensitive and label-free detection of uranyl ions (UO₂²⁺) has been proposed, which is easy to construct and does not need to be combined with a hydrogel. The PC film is not pH-sensitive because it is obtained by the self-assembly of methyl methacrylate-acrylonitrile co-polymeric nanospheres (PMMA-AN). These nanospheres were modified with amidoxime groups, which have a good coordination ability with UO₂²⁺. The bindings between nanospheres and UO₂²⁺ change the refractive index and disturb the face-centered cubic structure of the film, which leads to a decrease in the diffraction peak intensity of the PC film. The sensor works in the concentration range of 10 pM to 100 μM for UO₂²⁺ determination and the decreased intensities of the diffraction peaks are linearly correlated with the logarithm of UO₂²⁺ concentration in the range from 1 nM to 100 μM. Moreover, the sensor shows good selectivity for UO₂²⁺ and can also perform the determination of UO₂²⁺ in a real sample. The responsive PC film sensor shows great potential in the label-free and ultrasensitive detection of UO₂²⁺.