The previous visual P300-speller mostly used two-dimensional presentation on computer screens, while virtual reality (VR) technology can present stimulus in a virtual three-dimension (3D) space. This study intended to explore the effect of 3D presentation of visual stimuli on P300-speller in VR. Four different presentation paradigms, including three 3D paradigms and one two-dimension (2D) paradigm, were adopted. These 3D paradigms formed 3D visual stimuli by presenting depth information in character intensifications dynamically. The features of event-related potentials (ERP) and the spelling performance were analyzed, and the results showed that the electroencephalograph (EEG) class discriminant and accuracy of the 3D paradigm were superior to the 2D paradigm. Especially the two paradigms, Different depth information Array Character Flash (DACF) paradigm and Same depth information Array Character Jump (SACJ) paradigm, were recommended.