Harmful fine-tuning attack poses a serious threat to the online fine-tuning service. Vaccine, a recent alignment-stage defense, applies uniform perturbation to all layers of embedding to make the model robust to the simulated embedding drift. However, applying layer-wise uniform perturbation may lead to excess perturbations for some particular safety-irrelevant layers, resulting in defense performance degradation and unnecessary memory consumption. To address this limitation, we propose Targeted Vaccine (T-Vaccine), a memory-efficient safety alignment method that applies perturbation to only selected layers of the model. T-Vaccine follows two core steps: First, it uses gradient norm as a statistical metric to identify the safety-critical layers. Second, instead of applying uniform perturbation across all layers, T-Vaccine only applies perturbation to the safety-critical layers while keeping other layers frozen during training. Results show that T-Vaccine outperforms Vaccine in terms of both defense effectiveness and resource efficiency. Comparison with other defense baselines, e.g., RepNoise and TAR also demonstrate the superiority of T-Vaccine. Notably, T-Vaccine is the first defense that can address harmful fine-tuning issues for a 7B pre-trained models trained on consumer GPUs with limited memory (e.g., RTX 4090). Our code is available at https://github.com/Lslland/T-Vaccine.