Ultrasound, a non-invasive stimulation method, has proved effective in neurostimulation. Previous studies have demonstrated that low-frequency ultrasound (less than 1 MHz) is preferable owing to better penetrability through tissue and skull. However, the large size of low-frequency transducers, which are used in ultrasound neurostimulation studies, makes it difficult to perform multiple-target neurostimulation, especially in small animals such as mice. In this paper, a proposed low-frequency ultrasound needle transducer based on the multi-layer lateral-mode coupling method with a miniature aperture of 0.6 mm × 0.6 mm and a thickness of 1.65 mm was designed and fabricated. The measured electrical impedance of the fabricated 8-layer lateral-mode PZT-5H ceramic was 50.76 Ω at a resonant frequency of 866 kHz. The -6 dB bandwidth of 8-layer lateral-mode transducer was 29% at a center frequency of 876 kHz. The maximum ultrasound peak pressure amplitude at 820 kHz reached approximately 300 kPa, 4-5 times higher than that of the single-layer thickness-mode transducer with 200 V input voltage. The ultrasound beam showed no attenuation and low shift through mouse skull. To verify the feasibility of using the needle transducer to perform multiple-target nerve stimulation in mice brains, we constructed an ultrasound stimulus system to simultaneously stimulate two areas (M2 and V1) of the mouse brain in vivo and detected the c-Fos expression by immunofluorescence to evaluate the effect of stimulation. The results showed that a high ultrasound peak pressure amplitude with this transducer configuration is useful for ultrasound neurostimulation and multiple-target stimulation in mice.