The absence of better biomarkers currently limits early diagnosis and treatment of triple-negative breast cancer (TNBC). Our previously published study reported that the cyclic-peptide SD01 exhibited specific binding to EphA2 (Ephrin type-A receptor 2) on TNBC. To develop a novel PET imaging agent, we prepared gallium-68 (Ga) labeled-DOTA-SD01 and evaluated its specificity and effectiveness through micro PET/CT imaging in a TNBC-bearing mouse model. SD01 and a control linear peptide YSA were conjugated to DOTA and subsequently labeled with Ga, obtaining Ga-DOTA-SD01 and Ga-DOTA-YSA. Both showed high radiochemical purity, stability, good hydrophilicity, and high binding affinity to 4T1 cells. Micro PET/CT imaging showed high radioactivity accumulation in tumors; SUV (mean standardized uptake value) of tumors in the group of Ga-DOTA-SD01 was 3.34 ± 0.25 and 2.65 ± 0.32 in the group of Ga-DOTA-YSA; T/NT ratios (target to non-target, SUV ratios of tumor to muscle) were 3.12 ± 0.06 and 2.77 ± 0.11 at 30 min, respectively ( < 0.05). The biodistribution study showed that tumor uptake % ID per g (percentage of injected dose per gram of tissue) in the group of Ga-DOTA-SD01 was 2.73 ± 0.34, and 1.77 ± 0.38 in the group of Ga-DOTA-YSA; T/NT ratios (radioactivity of tumor to muscle) were 3.55 ± 0.12 and 3.05 ± 0.10 for both groups at 30 min, respectively ( < 0.05). All these suggest that Ga-DOTA-SD01 may act as a better novel PET imaging agent for EphA2 positive tumors, such as TNBC.